Global Proteomic Screening Of Random Bead Arrays Using Mass Spectrometry Imaging

ABSTRACT

Methods for proteomic screening on random protein-bead arrays by mass spec is described. Photocleavable mass tags are utilized to code a protein library (bait molecules) displayed on beads randomly arrayed in an array substrate. A library of probes (prey) can be mixed with the protein-bead array to query the array. Because mass spec can detect multiple mass tags, it is possible to rapidly identify all of the interactions resulting from this mixing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to co-pending U.S. ProvisionalApplication Ser. No. 61/359,964 filed Jun. 30, 2010, herein incorporatedby reference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to biology, molecular biology, biochemistry, cellbiology, biomedicine, biomarkers and clinical diagnostics; proteomics,reverse proteomics and mass spectrometry; bio-molecular arrays,microarrays, bead-arrays, and bead-displays; multiplexed assays andbio-assays; and label-free bio-molecular detection. More specifically,the invention relates to detecting or imaging molecules or compounds onindividual beads or particles using mass spectrometry as applied to theaforementioned fields.

BACKGROUND OF THE INVENTION A. Multiplexing in Bio-Molecular Detection

There is a continuing realization that the complexities of biologicalsystems can neither be fully understood nor harnessed by taking singlemeasurements or determinations in. a single assay or experimentalprocess. As a result, the biological, biotechnological and biomedicalfields continue to move towards multiplexing, that is, the capability toperform simultaneous, multiple determinations in a single assay orexperimental process [U.S. Pat. No. 5,981,180].

A.1 Multiplexing with Planar Bio-Molecular Arrays and Microarrays:

One important advancement in multiplexed biological experimentation orbio-assays has been through the introduction of microarrays, orso-called “chips”, which consist normally of an ordered and addressablearray of tens of thousands of microscopic spots or “features”, usuallyrobotically printed [MacBeath and Schreiber (2000) Science 289: 1760-3;Auburn, Kreil, Meadows, Fischer, Matilla and Russell (2005) TrendsBiotechnol 23: 374-9] to a single planar substrate typically thedimensions of a standard microscope slide; each feature containing aunique “bait” molecule, most commonly oligonucleotides or proteins,including antibodies. The entire chip is typically treated with a simpleor complex biological sample or complex mixture of molecules and thebait molecules on the chip bind or interact with the analyte(s) in thesample. These analytes are sometimes termed prey molecules. It is alsoto be understood that prey molecules may constitute biomarkers in acomplex mixture or molecules in a solution whose interaction with thebait molecules is to be determined. In some cases, the bound analyte(s)are measured, in others, the effects of analyte (prey molecule)interaction with the bait molecules is measured, for example, whereby ananalyte, such as a protein kinase, enzymatically modifies a baitmolecule on the chip (in this case, phosphorylation of the bait by theprotein kinase). The chip is then scanned or imaged in order to detectthese interactions, usually through a variety of fluorescence “reporter”methods. Alternatively, other reporters such as radioisotopes have beenused [MacBeath and Schreiber (2000) Science 289: 1760-3.]. Furthermore,label-free methods such as surface plasmon resonance [Boozer, Kim, Cong,Guan and Londergan (2006) Curr Opin Biotechnol 17: 400-5] or massspectrometry [Gabriel, Ziaugra and Tabbaa (2009) Curr Protoc Hum GenetChapter 2: Unit 2 12] are also possible. In some cases, a “probe” isused to assist in detection, for example, a substance that binds abait-bound analyte, such as antibody, and is capable if being detected(e.g. labeled).

DNA microarrays [Schena, Shalon, Davis and Brown (1995) Science 270:467-70] are now widely used and accepted by the scientific community,most commonly used for multiplexed, “genome-wide” analysis of the entireexpressed mRNA complement of a cell, tissue or other biological sample.In this case, the microarray features are oligonucleotide bait moleculesthat bind complementary mRNA or cDNA from a complex biological sample.Other examples of DNA microarray applications include single nucleotidepolymorphism (SNP) genotyping and mutation analysis [Bier, vonNickisch-Rosenegk, Ehrentreich-Forster, Reiss, Henkel, Strehlow andAndresen (2008) Adv Biochem Eng Biotechnol 109: 433-53], copy numbervariation (CNV) [Yau and Holmes (2008) Cytogenet Genome Res 123: 307-12]and chromatin immunoprecipitation (ChIP) analyses (so calledChIP-on-Chip) [Muro, McCann, Rudnicki and Andrade-Navarro (2009) MethodsMol Biol 567: 145-54].

Likewise, protein microarrays [MacBeath and Schreiber (2000) Science289: 1760-3; Zhu, Klemic et al. (2000) Nat Genet 26: 283-9] are rapidlygaining popularity. The most widely used forms can be classified as: i)“capture chips”, whereby the features/probes on the microarraycorrespond to affinity capture elements, usually antibodies, used toquantify the level of various analytes in a complex biological sample orii) “interaction chips”, whereby protein features/probes on themicroarray, usually recombinant proteins, are used to measurebiologically relevant interactions, such as protein-protein orprotein-drug interactions or enzymatic/chemical modification of theprotein probes on the microarray.

A.2 Multiplexing with Suspension Arrays and Bead-Arrays:

While fixed addressable/ordered microarrays are one mode of bio-assaymultiplexing, bead or particle based multiplexing, sometimes referred toas “suspension arrays” or “bead arrays” affords many advantages [Matherand Kelso (2009) Cytometry A]. Advantages include for example: i)“solution-phase” or homogeneous reaction and binding kinetics;ii),elimination of the need for mechanical printing and drying of themicroarray probes, a procedure which is subject to failure such asmisprinting and is also known to damage delicate biomolecules such asproteins; iii) increased density (diversity) of the bead-based probelibraries due to the facile production of very small, e.g. sub-microndiameter, beads or particles thereby allowing several orders ofmagnitude higher multiplexing levels compared to 2-dimensional planarmicroarrays. For example, bead-arrays in etched microscopic hexagonallypacked wells can reach densities of 10⁹/cm² [Michael, Taylor, Schultzand Walt (1998) Anal Chem 70: 1242-8] versus 10⁴/cm² for mechanicallyprinted spots on 2-dimensional planar microarrays [Mathur and Kelso(2009) Cytometry A]; iv) the ability to physically isolatesub-populations of beads or particles based on specific properties; andv) more facile use of 3-dimensional hydrated solid-matrices for probeattachment, such as commonly available porous agarose beads, that offera more bio-compatible surface as well as higher probe binding capacitiesthan planar microarrays.

A.3 Mainstream Light Based Bead Coding Methods:

Methods of encoding and decoding beads or particles are required inorder to facilitate the aforementioned bead-based multiplex bio-assaysand exploit their many advantages. Prominent commercial examples ofmultiplex bead/particle platforms include the xMAP® technology ofLuminex Corporation (Austin, Tex.), which uses beads encoded withfluorescent dyes and readout on a flow cytometry based platform [Fulton,McDade, Smith, Kienker and Kettman (1997) Clin Chem 43: 1749-56], andVeraCode technology of Illumina Incorporated (San Diego, Calif.), whichuses microscopic cylindrical glass microbeads encoded with digitalholographic “bar codes”[Lin, Yeakley, McDaniel and Shen (2009) MethodsMol Biol 496: 129-42]. Other examples of light based or spectral codingof beads or particles have been reported. For example, in 2001 Han etal. predicted that more than 40,000 distinct codes should be possible,for example when fluorescent quantum dot nanocrystals are permanentlyembedded in beads at different ratios of color and intensity [Han, Gao,Su and Nie (2001) Nat Biotechnol 19: 631-5]. However, in practice, suchmethods to date have not exceed a few hundred codes [Mathur and Kelso(2009) Cytometry A]. Other optical encoding techniques, for exampleemploying lithography (Multifunctional encoded particles forhigh-throughput biomolecule analysis. (Pregibon D C, Toner M, Doyle P S.Science. 2007 Mar. 9; 315(5817):1393-6) or fluorescence of rare earthelements (Parallel Synthesis Technologies Inc; www.parallume.com) canpotentially generate hundreds of thousands of unique codes but have notyet demonstrated their commercial viability.

A.4 Mass Coding of Beads or Particles and Mass Spectrometry:

Mass spectrometry (MS) has been used extensively as an analyticaltechnique in biotechnology for a variety of applications includingproteomics, biomarker discovery, genomic analysis and clinical assays[Koster, H., Tang, K., Fu, D. J., Braun, A., van den Boom, D., Smith, C.L., Cotter, R. I., and Cantor, C. R. (1996) Nat Biotechnol 14,1123-1128]. Very high throughputs are obtained because separation timesare measured in microseconds rather than minutes or hours compared toconventional methods such as gel electrophoresis [Ross, P., Hall, L.,Smirnov, I., and Haff, L. (1998) Nat Biotechnol 16, 1347-1351].Additional information, such as protein sequence and modificationsoccurring at specific residues is also possible using tandem massspectrometry (MS/MS) [Washburn, Wolters and Yates (2001) Nat Biotechnol19: 242-7].

The extremely high resolution and mass accuracy of mass spectrometryoffers the potential to greatly increase the number of possible uniqueidentification “codes” for beads or particles. Indeed, in the field ofproteomics, those skilled in the art will recognize that massspectrometry is a critical tool used in the identification of proteins.In a typical proteomics scenario, proteins are digested, such as byprotease, and identification of the protein achieved by one of two waysusing mass spectrometry a) mass fingerprinting—for a single species ofdigested protein (such as that isolated by two-dimensional gelelectrophoresis prior to digestion), the pattern of masses of thedaughter peptide fragments (“fingerprint”) can be sufficient foridentification or b) tandem mass spectrometry based sequencing of even asingle daughter peptide fragment can be sufficient for identification(e.g. see [Washburn, Wolters et al. (2001) Nat Biotechnol 19: 242-7]).

Not surprisingly, for multiplexed bio-assays, mass spectrometry has beenused in conjunction with so called “mass tags” as coding agents, forexample peptide mass tags [Olejnik, Ludemann, Krzymanska-Olejnik,Berkenkamp, Hillenkamp and Rothschild (1999) Nucleic Acids Res 27:4626-31] and oligonucleotide mass tags [Zhang, Kasif and Cantor (2007)Proc Natl Acad Sci USA 104: 3061-6] have been reported. U.S. Pat. No.6,218,530, “Compounds and Methods for Detecting Biomolecules” herebyspecifically incorporated into this application (has peptide mass tagsin specifications). Previously, mass tags have been used to code beadlibraries and detected by mass spectrometry, particularly in the fieldsof combinatorial chemistry and solid-phase organic synthesis. However,in these studies the detection was performed after elution of the masstags and not directly from individual beads or from arrays of particlesusing mass spectrometric imaging techniques. The elution was achieved byeither prolonged exposure to acid or UV irradiation [J. Comb. Chem.2003, 5, 125-137 “High-Throughput One-Bead-One-Compound Approach toPeptide-Encoded Combinatorial Libraries: MALDI-MS Analysis of SingleTentaGel Beads” Andreas H. Franz, Ruiwu Liu, Aimin Song, Kit S. Lam, andCarlito B. Lebrilla; Anal. Chem. 2007, 79, 7275-7285 “Method forScreening and MALDI-TOF MS Sequencing of Encoded CombinatorialLibraries” Bi-Huang Hu, Marsha Ritter Jones, and Phillip B. Messersmith]or alternatively, in the case of peptides attached to beads throughhydrophobic or antibody-mediated interactions, simply by the addition ofMALDI matrix [Anal Chem. 2004 Jul. 15; 76(14):4082-92. “Development of aprotein chip: a MS-based method for quantitation of protein expressionand modification levels using an immunoaffinity approach”. Warren E N,Elms P J, Parker C E, Borchers C H.; Anal. Chem. 2005, 77, 1580-1587“Monitoring Activity-Dependent Peptide Release from the CNS UsingSingle-Bead Solid-Phase Extraction and MALDI TOF MS” Detection Nathan G.Hatcher, Timothy A. Richmond, Stanislav S. Rubakhin, and Jonathan V.Sweedler]. Several studies have also reported direct detection of masstags on beads and even examined their distribution within individualbeads using secondary ion mass-spectrometry (SIMS) [Comb Chem HighThroughput Screen. 2001 June; 4(4):363-73. “Mass spectrometry andcombinatorial chemistry: new approaches for direct support-boundcompound identification”. Enjalbal C, Maux D, Martinez J, Combarieu R,Aubagnac J L]. The SIMS technique provides high lateral resolution downto sub-micron range, but unlike MALDI MS generates only small ions (MWbelow 400 Da) and is therefore not suitable for proteomic or nucleotideanalysis. Importantly, all of the above studies do not describe MALDI-MSon individual beads or arrays of individual beads.

Mass spectrometry scanning or imaging can facilitate in situ detectionof mass tags directly from individually resolved beads and be used todecode the bead for rapid identification of other molecules (e.g. baitand prey) directly or indirectly bound to the bead. In one embodiment,this is done by mass-imaging with a Matrix Assisted Laser DesorptionIonization Time of Flight (MALDI-TOF) mass spectrometer. For example,beads or particles are deposited onto a surface which is then scannedwith the laser beam of the MALDI-TOF mass spectrometer, and a mass-imageis created of the bead “array” using the peak intensity at themass/charge ratio corresponding to that of the target compound (e.g.mass tag). Since the Nd—Yag laser beam used for MALDI-TOF massspectrometry is diffraction limited, it can be focused to less than 1micron, much smaller than the diameter of micro-beads (5-100 microns)commonly used for bio-assays. Typical beads used in bio-assays rangefrom porous cross-linked agarose beads, to solid paramagnetic beads(often with polymeric shell), silica beads and plastic polymer beadssuch as polystyrene polymers or co-polymers. Pre-cursor beads often havesurface chemistries (e.g. binding agents) to allow attachment of “bait”molecules or compounds needed for various bio-assays. Common beadsurface chemistries include chemically reactive groups, such asaldehyde, epoxy or succinimidyl esters, or molecular handles such asamine, sulfhydryl or carboxyl moieties typically used in conjunctionwith chemical cross-linkers. Passive adsorption of protein or nucleicacid based molecules for example, is also possible, typically viahydrophobic and/or ionic interactions with surface modifications on thebeads. All of these chemical groups are can potentially serve as bindingagents for bait molecules or for other molecules such as mass tags. Inaddition, bioreactive molecules bound to the surface of beads such asantibodies can also serve as binding agents for bait molecules or forother molecules such as mass tags.

The ability to perform mass spectrometry based scanning and mass-imagingof beads or particles, as described in this patent, opens the door fordramatic improvements in bio-assay multiplexing capabilities, with thepotential for millions of codes and facilitating multiplexing both atthe level of encoding beads or particles for identification as well asat the level of encoding the bio-molecular probes (sometimes termed preymolecules) present in samples or complex mixtures used to query thebeads (e.g. beads which may contain various “bait” molecules such asrecombinant proteins or antibodies for example). It is to be understoodthat in this invention biomarkers also in complex mixtures alsoconstitute prey molecules.

B. Proteomics: Applications of Large-Scale Multiplexing in Bio-MolecularDetection B.1 Proteomics:

The “central dogma”, first proposed by Francis Crick in the 1950's,describes the process by which the genetic material in cells, DNA, isconverted to the cell's machinery, proteins. Now, after over 50 years,science has succeeded in decoding the DNA contained in the approximately25,000 genes in the human genome [Consortium (2004) Nature 431: 931-45;Stein (2004) Nature 431: 915-6]. While this accomplishment represents amajor success for this first “Manhattan-scale” project in biology, amuch more ambitious goal is emerging for the post-genome era. This goalis to analyze the entire protein complement of the genome, firstreferred to as the proteome [Wasinger, Cordwell et al. (1995)Electrophoresis 16: 1090-4] in 1994 by Marc Wilkins and Keith Williamsof the Macquarie University Center for Analytical Biotechnology (MUCAB)in Sydney, Australia. While the proteome is the entire expressedcomplement of a genome, those skilled in the art will recognize thatproteomics involves the global analysis of entire proteomes in a singleexperimental process (i.e. multiplexed analysis).

In principle, just as whole genomes are now more rapidly analyzed usingnext-generation massively parallel DNA sequencing [Shaffer (2007) NatBiotechnol 25: 149], equally powerful methods are needed for proteomicsscreening. The potential benefits of such screening for improving humanhealth are enormous, since understanding the basis of diseases dependscritically on understanding the machinery of the cell, i.e. proteinsexpressed by the genome.

In general, as detailed below, proteomics can be divided into twocategories, that is, “classical” (forward) proteomics and reverseproteomics:

B.2 Classical Proteomics:

In this “forward” proteomics model (see below for reverse), one beginswith an entire proteome which is then linked or mapped to the genomeduring a protein analysis and identification process [Wasinger, Cordwellet al. (1995) Electrophoresis 16: 1090-4; Celis, Ostergaard, Jensen,Gromova, Rasmussen and Gromov (1998) FEBS Lett 430: 64-72]. Theproteomes are typically first extracted from complex biological samplessuch as cells, tissues or biological fluids for downstream multiplexedanalysis. Classical proteomics methods were originally configured asseparation and analysis of the entire extracted proteomes bytwo-dimensional gel electrophoresis, followed by identification ofproteins excised from the gel by mass spectrometry [Wasinger, Cordwellet al. {1995) Electrophoresis 16: 1090-4], although identification byantibody recognition or protein sequencing has also been used [Celis,Ostergaard et al. (1998) EBBS Lett 430: 64-72]. Such approaches are nowjoined by “gel-free” or “shot-gun” proteomics methods that avoid the useof two-dimensional electrophoresis. These methods are usually based onfragmentation of the entire proteome into peptides, peptidepre-fractionation (typically by multi-dimensional high resolution liquidchromatography) and analysis/identification by mass spectrometry (seefor example [Patton, Schulenberg and Steinberg (2002) Curr OpinBiotechnol 13: 321-8] and [Washburn, Wolters et al. (2001) NatBiotechnol 19: 242-7]).

The most common application of classical proteomics is in differentialprotein expression profiling, where protein expression levels in acontrol sample are compared to that of a test sample in order toidentify proteins of interest (e.g. disease associated) on aproteome-wide scale. However, variants have also been used, such asdifferential analysis of protein modification, for example,post-translational modifications such as phosphorylation (e.g. [Takano,Otani, Sakai, Kadoyama, Matsuyama, Matsumoto, Takenokuchi, Sumida andTaniguchi (2009) Neuroreport 20: 1648-53]).

In another embodiment of “forward” proteomics, extracted proteomes aremapped to the genome through specific recognition by affinity elements.In practice, this is usually achieved in multiplex format using antibodyor “capture” arrays/microarrays, by capture and quantification ofproteins from a complex mixture (proteome) using specific antibodies(affinity elements) printed to the array surface [Borrebaeck and Wingren(2009) J Proteomics 72: 928-35]. These techniques are also mosttypically used for proteome-wide protein expression profiling.

B.3 Interaction Based Proteomics:

Expanding beyond the protein expression profiling that is typical ofclassical proteomics, an ideal proteomic screen would provide all theinformation necessary to identify all possible interactions between theM proteins in the proteome with N other molecules (e.g. proteome,nucleome and metabolome), in an M×N interaction matrix. It is to beunderstood in this case that there are M probe molecules and N preymolecules. In the case of a full probing of protein-protein interactionsin a library of M proteins which potentially serve as both bait andprey, this matrix would have M² elements. While a variety of techniquesexist to measure such interactions, they are usually based on screeningthe interaction of a single probe molecule against a set of othermolecules, essentially providing only one row in the interaction matrix.One such extensively used method involves tandem affinity purification(TAP) of expressed target proteins and identification of interactingproteins by tandem mass spectrometry (MS/MS) [Collins and Choudhary(2008) Curr Opin Biotechnol 19: 324-30]. In contrast, yeast two-hybridmethods, based on in vivo screening of a protein library against asingle protein or against an another library, can specify all theelements of an M×N matrix. However, this technique requires thescreening and partial sequencing of M×N from different cell colonies,provides only binary information (e.g. interaction occurs or does notoccur) and has as high as a 50% false-positive/negative rate [Suter,Kittanakoni and Stagljar (2008) Curr Opin Biotechnol 19: 316-23].

B.4 Reverse Proteomics and Proteome Arrays:

Reverse proteomics represents an important tool in interaction basedproteomic screening. In this reverse format, a set of genes or a genelibrary (a “genome”) is used to generate (synthesize) a proteome forstudy in a multiplexed format [Rual, Hirozane-Kishikawa at al. (2004)Genome Res 14: 2128-35]. In principle, the entire human proteome couldbe generated from the human genome and each protein analyzed for itsdifferent properties (e.g. protein interactions). While such a globaltranslation of the human genome has never been achieved, even a limitedset of genes can yield valuable information.

One widely used example of reverse proteomics is proteome microarrays(an “interaction chip”), that is, microarrays of purified recombinantproteins corresponding to an entire proteome (full expressed complementof a genome) or a large fraction thereof. Proteome microarrays arecurrently being used for various applications including mappingprotein-protein interactions for elucidating cellular pathways [MacBeathand Schreiber (2000) Science 289: 1760-3; Zhu, Bilgin et al. (2001)Science 293: 2101-5; Ramachandran, Hainsworth, Bhullar, Eisenstein,Rosen, Lau, Walter and LaBaer (2004) Science 305: 86-90], determiningprotein-small molecule interactions including with drug compounds[MacBeath and Schreiber (2000) Science 289: 1760-3.], analysis ofenzymatic activities such as kinase substrate preference [MacBeath andSchreiber (2000) Science 289: 1760-3; Zhu, Klemic et al. (2000) NatGenet. 26: 283-9], evaluating antibody specificity [Michaud, Salcius,Zhou, Bangham, Bonin, Guo, Snyder, Predki and Schweitzer (2003) NatBiotechnol 21: 1509-12] and biomarker discovery [Sheridan (2005) NatBiotechnol 23: 3-4], such as in the discovery of novel autoantigens inautoimmune diseases as well as cancers [Robinson, DiGennaro et al.(2002) Nat Med 8: 295-301; Robinson, Fontoura et al. (2003) NatBiotechnol 21: 1033-9; Hudson, Pozdnyakova, Haines, Mor and Snyder(2007) Proc Natl Acad Sci USA 104: 17494-9; Babel, Barderas,Diaz-Uriarte, Martinez-Torrecuadrada, Sanchez-Carbayo and Casal (2009)Mol Cell Proteomics 8: 2382-95].

B.4.1 Conventional Cell-Derived Recombinant Proteome Arrays

Unlike DNA microarrays [Schulze and Downward (2001) Nat Cell Biol 3:E190-5.], where oligonucleotide probes for each expressed gene can bereadily synthesized, creating a purified set of arrayed cellularproteins or antibodies (as shown in FIG. S02) is significantly moredifficult. This process involves the production of tens of thousands ofrecombinant proteins using gene cloning, in vivo cellular expression,protein purification and mechanical microarray printing [MacBeath andSchreiber (2000) Science 289: 1760-3; Zhu, Bilgin et al. (2001) Science293: 2101-5]. These methods are often slow, labor intensive and heavilydependent on highly specialized robotics, such as serial microarrayprinters/spotters which are expensive and subject to failure; the netresult is prohibitively expensive protein arrays of limited density andlimited scalability for larger protein content.

For example, Invitrogen has introduced the first commercial humanproteome microarray [Zhu, Bilgin et al. (2001) Science 293: 2101-5]. Itcontains roughly 9,000 distinct proteins, representing a small fractionof the predicted human proteome [Melton (2004) Nature 429: 101-7], at acost of $1,725/microarray (˜$0.2/protein). While costs may come down asmore efficient methods of protein production and isolation areintroduced, fundamental limitations still remain—namely the need forindividually cloning each gene, individually expressing each protein incells, separate isolation of each protein, mechanical microarrayprinting of the proteins, stability of the protein stocks or arraysderived from them and difficulties in expressing proteins that are toxicto the host cell. Furthermore, at ˜20,000 spots total (replicates andcontrols), Invitrogen's microarray capacity is nearly at it's maximumsince protein arrays are not compatible with the photolithography usedin DNA microarrays to create smaller and more densely packed spots. A2005 review in Nature Biotechnology finds that “Invitrogen's recentlaunch of what it billed as the world's first commercially availablehuman protein microarray may, paradoxically, signal the abandonment, fornow at least, of the grand ambition of characterizing the entire humanproteome using a single chip” [Sheridan (2005) Nat Biotechnol 23: 3-4].Instead, the report contends, protein chip companies are focusing onselected microarray content (smaller protein subsets), custom tailoredto specific applications.

An additional limitation of the current generation of proteomic arraysis the intrinsic low sensitivity and high noise which impedes biomarkerdiscovery from clinical samples. Part of this problem derives from thelow replicate number (duplicate) for each protein represented and thevariability of spot printing and subsequent readout. In particular,conventional arrays are fabricated by printing and drying thousands of100 micron protein spots on a flat surface (e.g. nitrocellulose film).The protein antibody interaction than occurs on top of this aggregatedprotein spot which is then again dried for read-out. For these reasons,the assay conditions are far from the ideal solution-phase functionalassays. Ideally, proteins need to be arrayed in small “reaction vessels”where the protein-antibody interaction occurs and is measured. However,this is difficult, if not impossible to achieve using conventionalprotein microarray technology.

B.4.2 Cell-Free Expression-Based Proteome Arrays

Until recently, relatively high costs and low yields have discouragedthe use of cell-free (in vitro) protein expression systems in the fieldof proteomics. However, recent improvements in this field hold greatpromise for solving many of the problems associated with conventionalproteomic arrays [Rothschild and Gite (1999) Curr Opin Biotechnol 10:64-70; He and Taussig (2001) Nucleic Acids Res 29: E73-3; Kawahashi,Doi, Takashima, Tsuda, Oishi, Oyama, Yonezawa, Miyamoto-Sato andYanagawa (2003) Proteomics 3: 1236-43; Ramachandran, Hainsworth et al.(2004) Science 305: 86-90; Gite, Lim and Rothschild (2006) Biotechnology& Genetic Engineering Reviews 22: 151-169]. Advantages and improvementsinclude: On-Demand Expression: Express specific proteins, on-demand,typically in <1 hr, even in eukaryotic (e.g. mammalian or insect)systems using a single facile reaction (e.g. Promega's batch mode rabbitreticulocyte or insect cell coupled transcription/translation system;Promega Corporation, Madison, Wis.). Recently, over 13,000 differentproteins from the human genome were expressed using an improvedcell-free wheat germ expression system demonstrating the feasibility ofusing cell-free techniques for a proteome factory [Goshima, Kawamura etal. (2008) Nat Methods 5: 1011-7]. High Yield: New “continuous exchange”cell-free (CECF) expression systems capable of mg/mL yields (e.g.Roche's Wheat Germ CECF; (Roche Applied Science, Indianapolis, Ind.)).Protein Compatibility: Often cellular systems cannot express proteinsdue to the cytotoxicity or interference with host cell physiology[Henrich, Lubitz and Plapp (1982) Mol Gen Genet 185: 493-7; Goff andGoldberg (1987) J Biol Chem 262: 4508-15; Nakano and Yamane (1998)Biotechnol Adv 16: 367-84; He and Taussig (2001) Nucleic Acids Res 29:E73-3; Endo and Sawasaki (2003) Biotechnol Adv 21: 695-713]. MembraneProteins: Normal expression of these proteins in cells is not easilycompatible with microarray technology since membrane proteins have to beisolated in detergent and reconstituted in model lipid bilayer systems.However, recently progress in cell-free protein techniques has made itpossible to incorporate membrane proteins in a single step intonanolipoparticles [Cappuccio, Blanchette et al. (2008) Mol CellProteomics 7: 2246-53; Katzen, Fletcher et al. (2008) J Proteome Res 7:3535-42; Cappuccio, Hinz et al. (2009) Methods Mol Biol 498: 273-96],small discoidal membranes mimicking the native membrane proteinenvironment. In addition, commercial kits such as the InvitrogenMembraneMAX™ cell-free protein expression kits are available(Invitrogen, Carlsbad, Calif.).

B.4.3 Application of Reverse Proteomics and Proteome Microarrays toAutoantigen Discovery in Cancers and Autoimmune Diseases Autoimmune:

More than 80 illnesses have been described that are associated withactivation of auto-reactive lymphocytes and the production ofautoantibodies directed against normal tissue or cellular components(autoantigens) [von Muhlen and Tan(1995) Semin Arthritis Rheum 24:323-58; Mellors (2002) 2005]. Collectively referred to as autoimmunediseases, they afflict an estimated 15-24 million people (at least 3-5%)in the U.S. and constitute a major economic and health burden [Jacobson,Gange, Rose and Graham (1997) Clin Immunol Immunopathol 84: 223-43]. Ahost of common diseases fall into this category including multiplesclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus(SLE), Sjögrens Syndrome (SjS), insulin-dependent diabetes (IDDM),myasthenia gravis (MG), psoriasis, scleroderma and primary biliarycirrhosis (PBC) [Mellors (2002) 2005].

The root causes of the immune dysfunction underpinning autoimmunedisease are still not well understood. Consequently, autoimmune diseasesgenerally remain difficult to diagnose based on clinical presentation,which typically involves a constellation of symptoms. The ability todetect serum autoantibodies greatly facilitates the diagnosis ofautoimmune diseases. In the past, patient serum was screened forautoantibodies by indirect immunofluorescence (IIF) using a human cellline (Hep-2) as substrate. In recent years, national clinicallaboratories have abandoned screening for autoantibodies by IIF and haveswitched to solid-phase assays. These assays, which include ELISAs andmultiplexed bead-based platform technologies (e.g. Luminex Corporation,Austin, Tex.), use a limited number of purified native or recombinantantigens to screen for autoantibodies (termed here bait molecules asopposed to serum antibodies which are termed prey molecules). Therationale for the change to solid-phase assays is that these tests canbe automated, significantly reducing labor costs. Furthermore, theseassays produce quantitative and hence objective results, as opposed tothe subjective nature of IIF.

The American College of Rheumatology recently convened an Ad Hoccommittee to investigate whether screening for autoantibodies usingsolid phase assays is equivalent to screening for these antibodies usingindirect immunofluorescence(http://www.rheumatology.org/publications/position/ana_position_stmt.pdf).After careful review of the available scientific literature, thecommittee determined that solid phase assays are not equivalent to IIF.The committee noted that the Hep-2 cell substrate contains more than 100clinically-relevant autoantigens. In contrast, solid phase assayscontain only a limited number of antigens. For example, the AtheNA™anti-nuclear autoantibody (ANA) assay (Zeus Scientific, Branchburg,N.J.) based on the Luminex platform, screens for antibodies directedagainst only 14 nuclear autoantigens. Because the Hep-2 cell substrateand indirect immunofluorescence detects a larger number ofautoantibodies, the committee concluded that solid phase assays, as theyexist today, can not be used as a substitute for IIF to screen forautoantibodies.

For example, the limitations of solid phase assays for the detection ofSLE-related autoantibodies were recently illustrated in a Case Recordpublished in the New England Journal of Medicine [Kroshinsky, Kay andNazarian (2009) N Engl J Med 361: 2166-76]. The appropriate diagnosis ofSLE in the patient was significantly delayed because autoantibodies werenot detected by the AtheNA™ Luminex assay. However, antinuclearantibodies were detected at high titer in the patient serum by IIF usingthe Hep-2 cell substrate,

Screening for autoantibodies by solid phase assays is significantlyfaster and more cost-effective than screening tests that rely on IIF.However, as described above, solid-phase assays contain far fewerautoantigens than are present in the Hep-2 cell substrate. Many of theautoantigens present in Hep-2 cells have not yet been characterized. Toimprove the sensitivity of solid phase assays, additionalclinically-relevant autoantigens will have to be identified, produced,purified and included in future solid phase assay kits.

Cancer:

In cancer, a growing body of evidence indicates [Chapman, Murray,McElveen, Sahin, Luxemburger, Tureci, Wiewrodt, Barnes and Robertson(2008) Thorax 63: 228-33] that autoantibodies form againsttumor-associated autoantigens (TAA) and that these autoantibodies arepresent even in the early stages of disease.

There have been numerous reports which demonstrate the importance of TAAdiscovery for an immunological approach to cancer diagnostics. Forexample, a recent study on TAA for non-small and small-cell lung cancer[Chapman, Murray et al. (2008) Thorax 63: 228-33] reported that at least1 antibody was detected out of a panel of 7 antigens in 76% of patientsstudied with 92% specificity. Since selection of the panel was basedonly on a small subset of proteins associated with cancer (p53, c-myc,HER2, NY-ESO-1, CAGE, MUC1 and GBU45) a more global proteomic approachis expected to lead to more sensitive and specific signatures. Forexample, in the case of heptocellular carcinoma (HCC), the use ofserological proteome analysis (SERPA) led to a panel of 6 antigens whichgave a sensitivity of 90% [Li, Chen, Yu, Li and Wang (2008) J ProteomeRes 7: 611-20]. In the case of ovarian cancer, over 50 putativeautoantigens involved in both a humoral and cell-mediated immuneresponse were identified using a proteomic mass spectrometric approach[Philip, Murthy, Krakover, Sinnathamby, Zerfass, Keller and Philip(2007) J Proteome Res 6: 2509-17].

Reverse Proteomics in Autoantigen Discovery:

Proteomics and proteome microarrays in particular are ideally suited forthe discovery of novel diagnostic autoantigen biomarkers for bothcancers and autoimmune diseases. Small volumes of patient blood, plasmaor serum samples are rapidly screened for autoantibodies, in unbiasedfashion, against a large fraction of the human proteome present on anaddressable chip in highly purified form. W. H. Robinson and P. J. Utzhave done extensive work in this field using medium density proteinarrays with a variety of autoimmune disorders. In addition to diagnosis,autoantigen biomarkers can be used for prognosis, disease staging and toassist in the development of tolerizing therapies [Robinson, DiGennaroet al. (2002) Nat Med 8: 295-301; Robinson, Garren, Utz and Steinman(2002) Clin Immunol 103: 7-12; Robinson, Steinman and Utz (2002)Arthritis Rheum 46: 885-93; Robinson, Fontoura et al. (2003) NatBiotechnol 21: 1033-9; Graham, Robinson, Steinman and Utz (2004)Autoimmunity 37: 269-72]. Partial proteome microarrays have also beenused for the discovery of TAA in colorectal cancer [Babel, Barderas etal. (2009) Mol Cell Proteomics 8: 2382-95], ovarian cancer [Hudson,Pozdnyakova et al. (2007) Proc Natl Acad Sci USA 104: 17494-9] andbreast cancer [Anderson, Ramachandran et al. (2008) J Proteome Res]. Thecurrent invention will greatly facilitate and accelerate these goals byproviding a faster, more flexible, less expensive and more robust methodof producing and assaying protein and proteome arrays and with a greaterscalability to true proteome-wide screening.

The following are examples of some of the many autoimmune diseases andcancers whose diagnosis, treatment and management may benefit fromproteomics based autoantigen discovery.

Examples of Autoimmune Diseases:

Primary Biliary Cirrhosis: PBC is an autoimmune disease characterized bythe gradual progressive destruction of intrahepatic biliary ductulesleading to hepatic fibrosis and liver failure (reviewed in [Kaplan(1996) N Engl J Med 335: 1570-80; Kaplan (2002) Gastroenterology 123:1392-4; Talwalkar and Lindor (2003) Lancet 362: 53-61]). It is the thirdleading indication for liver transplantation. Diagnosis of PBC iscurrently achieved by abnormal liver function tests, anti-mitochondrialantibodies (AMAs) and characteristic histological findings in a liverbiopsy specimen [Yang, Yu, Nakajima, Neuberg, Lindor and Bloch (2004)Clin Gastroenterol Hepatol 2: 1116-22]. However, initial PBC diagnosisis often missed because of the many vague and diffuse presentingsymptoms which are characteristic of many other autoimmune diseases[Bloch, Yu, Yang, Graeme-Cook, Lindor, Viswanathan, Bloch and Nakajima(2005) J Rheumatol 32: 477-83]. Although AMAs are a sensitive andspecific marker for this disease, the test may not be ordered in manypatients, especially when the patient presents with vague symptoms ofjoint discomfort. In addition, even when AMAs are present, their titeris highly variable and the titer does not predict disease severity orprognosis [Leung, Coppel, Ansari, Munoz and Gershwin (1997) Semin LiverDis 17: 61-9].

Systemic Lupus Erythematosus: Systemic lupus erythematosus (SLE) is achronic and potentially life-threatening autoimmune diseasecharacterized by multiple organ involvement [Sherer, Gorstein, Fritzlerand Shoenfeld (2004) Semin Arthritis Rheum 34: 501-37]. SLE afflicts300,000 to 1.5 million people in the U.S., with 16,000 new cases/year[2009; Ward (2004) J Womens Health (Larchmt) 13: 713-8; Chakravarty,Bush, Manzi, Clarke and Ward (2007) Arthritis Rheum 56: 2092-4]. SLEaffects primarily women in their child-bearing years, and is 9-fold moreprevalent in women than men. The 10 year survival rate of this diseaseis 80-90%, with approximately 1,300 deaths per year. During 1979-1998,the annual number of deaths from lupus rose from 879 to 1,406 [(2002)MMWR Morb Mortal Wkly Rep 51: 371-4].

For the past several decades, indirect immunofluorescence (IIF),especially of the nucleus, has been the method of choice by physiciansfor the detection of autoantibodies present in the serum of autoimmunepatients with SLE. Importantly, it remains the gold standard foranti-nuclear autoantibody (ANA) testing, including for SLE. Patientserum is serial diluted in two-fold increments and allowed to bind to aHEp-2 liver cell substrate on a microscope slide, which is thenfluorescently stained to detect bound autoantibodies and examined underthe microscope by a trained technician to identify the cellular stainingpatterns. However, this assay is problematic, as it is difficult tostandardize owing to variations in the substrate and fixation process,variations in the microscopy apparatus, and due to the highly subjectiveinterpretation of results [Jaskowski, Schroder, Martins, Mouritsen,Litwin and Hill (1996) Am J Clin Pathol 105: 468-73]. Furthermore, thisapproach is slow, laborious and not amenable to high throughputautomation [Ulvestad, Kanestrom, Madland, Thomassen, Haga and Vollset(2000) Scand J Immunol 52: 309-15]. This lack of throughput iscompounded by the fact that the diffuse presenting symptoms of SLEcauses doctors to often indiscriminately order IIF ANA tests, wastingprecious bandwidth [Suresh (2007) Br J Hosp Med (Lond) 68: 538-41].

Sjögren's Syndrome: Sjogren's (pSjS) is an autoimmune diseasecharacterized by chronic inflammation of the lacrimal and salivaryglands, resulting in the hallmark symptoms of dry eyes and mouth. Itconsidered the second most common autoimmune disease next to rheumatoidarthritis, however, most cases remain undiagnosed [Al-Hashimi (2007)Womens Health (Lond Engl) 3: 107-22]. The disease is differentiatedbetween primary and secondary Sjogren's (pSjS and sSjS), whereby glandinflammation does not or does occur in the presence of anotherconnective tissue disease, such as rheumatoid arthritis, systemic lupuserythematosus, primary biliary cirrhosis or scleroderma [Vitali,Bombardieri et al. (2002) Ann Rheum Dis 61: 554-8; Manoussakis (2004)Orphanet encyclopedia]. It is estimated that pSjS affects 1 to 4 millionpeople in the United States. The disease affects predominantly women(90% of SjS patients) in the post-menopausal years (40-50), althoughpeople of any age can develop the disease [Pillemer, Matteson,Jacobsson, Martens, Melton, O'Fallon and Fox (2001) Mayo Clin Proc 76:593-9; Manoussakis (2004) Orphanet encyclopedia; Alamanos, Tsifetaki,Voulgari, Venetsanopoulou, Siozos and Drosos (2006) Rheumatology(Oxford) 45: 187-91]. Misdiagnosis/under-diagnosis is primarily due tothe wide range of often vague clinical manifestations which overlap witha broad spectrum of other autoimmune disorders. While ANAs directedagainst the Ro/La RNP complex (SSA 52 kDa Ro, SSA 60 kDa Ro and SSB La)are the most common autoantibodies in SjS, they are also present inother autoimmune diseases, especially SLE [Mahler (2007) CurrentRheumatology Reviews 3: 67-78].

Example of Cancer Diseases:

There exists an urgent need to develop an effective non-invasive methodof detecting colorectal cancer (CRC), the second leading cause of cancerdeaths in the U.S and Western world. The American Cancer Societyestimates that there will be approximately 150,000 new cases ofcolorectal cancer (CRC) and 56,000 CRC related deaths per year. Thelife-time risk of colorectal adenocarcinoma is 6%, with it risingsteeply at ages over 60 [Davies, Miller and Coleman (2005) Nat RevCancer 5: 199-209]. Such non-invasive testing, if instituted for a largesegment of the population, could result in a dramatic reduction in themortality due to this disease. The American Cancer Society recommendsthat individuals over the age of fifty with normal risk be screened at1-5 year intervals using one or more of the current methods for earlyCRC detection, which include the fecal occult-blood test (FOBT) andendoscopic colorectal examination (colonoscopy). However, these methodsare of limited effectiveness, compliance and/or capacity to handlepopulation-wide screening.

In contrast, as described above, TAA hold significant promise for earlynon-invasive diagnosis of cancers such as CRC, especially if a panel ofTAA with high specificity could be developed. However, relatively fewerTAA have been identified and validated thus far for CRC compared toother cancers, such as ovarian and lung (see above). In one study, theuse of SEREX (serological identification of antigens by recombinantexpression cloning) resulted in the identification of 8 differentpotential clones for TAA, three of which (C210RF2, EPRS and NAP1L1) werefound mainly in cancer patients' sera [Line, Slucka, Stengrevics, Him,Li and Rees (2002) Cancer Immunol Immunother 51: 574-82]. WT1, which hasbeen shown to be overexpressed, stimulates cytotoxic T-cells making it acandidate for anti-CRC-vaccine development [Koesters, Linnebacher, Coy,Gerrnann, Schwitalle, Findeisen and von Knebel Doeberitz (2004) Int JCancer 109: 385-92]. Other TAA associated with CRC include colorectaltumor-associated antigen-1 (COA-1) [Maccalli, Li, El-Gamil, Rosenbergand Robbins (2003) Cancer Res 63: 6735-43], tumor-associated antigen90K/Mac-2-binding protein [Ulmer, Keeler, Loh, Chibbar, Torlakovic,Andre, Gabius and Laferte (2006) J Cell Biochem 98: 1351-66] andtumor-associated antigen TLP [Guadagni, Graziano, Roselli, Mariotti,Bernard, Sinibaldi-Vallebona, Rasi and Garaci (1999) Am J Pathol 154:993-9].

SUMMARY OF THE INVENTION A New Approach to Proteomics

One embodiment of this invention provides a novel, rapid andquantitative approach for global proteomic screening based on MatrixAssisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF MS) scanning/imaging performed on a random protein-beadarray. This approach, termed by us as bead-based global proteomicscreening (Bead-GPS), utilizes photocleavable mass tags to code both aprotein library (“bait” molecules) displayed on individual beadsrandomly arrayed in an array substrate such as a pico-well plate.Bead-GPS also uses a library of probe (“prey”) molecules such asproteins, nucleic acids and even complex biological samples such asserum from patients containing serum antibodies or cell lysates, whichare mixed with (i.e. used to query) the protein-bead array. Because wehave shown that MALDI-TOF MS can detect multiple mass tags from anindividual bead that has both the bait (e.g. protein) and probe (prey)molecules attached, it is possible to rapidly identify all theinteractions which occur among the millions of possible interactions inthe proteome. In addition, the ability to perform a fluorescent scan,either before or after MALDI-TOF MS scanning or imaging , on the samerandom bead-protein array (FIG. S03), provides additional informationabout the position of beads in the array displaying positiveinteractions between bait and prey and also information about thestrength of these interactions.

An additional embodiment of this invention involves rapid scanning of arandom array of beads for potential beads which exhibit specificinteract of the bait molecule residing on the bead and attached througha binding agent (e.g. bait molecule is a specific autoantigen bound tobead surface through an antibody binding agent) with a prey molecule(e.g. autoantibodies from a patient's blood) which interacts with saidbait molecule. Beads displaying positive interactions betweenautoantigens and autoantibodies are first identified on the basis ofdetection of fluorescence from a fluorophore which is directly orindirectly linked to the prey molecule through a binding agent and thenthe identity of the bait molecules residing on the bead determined bydecoding the identity of bead using a coding agent such as a mass tag.Two different methods of decoding positive beads (e.g. positive-hits) inthe bead array are based on: i) MALDI-TOF scanning/imaging of individualbeads, containing mass tags which in some cases are photocleavable (PC)mass tags, the beads being randomly arrayed on a specially designedpica-well slide containing ½-million wells in the dimensions of astandard microscope slide or Massively parallel RT-PCR orhybridization-based DNA/RNA microarray using photocleavable nucleic acid-tags (such as photocleavable DNA-tags). In either case, the aqueousenvironment provided for interaction between bait and prey (probe), thehigh number of replicate beads for each bead species in the totallibrary, plus the higher binding capacity per bead compared toconventional microarrays provides significant improvements in thebiomarker discovery process compared to conventional methods.

Bead-GPS can be readily adapted to a number of useful applications inproteomics, biomarker discovery and bio-molecular profiling. Thisincludes:

High Throughput Screening of a Drug Library Against a Proteomic Library:It is of great value in the pharmaceutical industry to screen thebinding of a small molecule compound library which can serve aspotential therapeutic drugs against a range of protein molecules orother biologically active molecules such as nucleic acids. In thisembodiment all compounds in the drug library (i.e. prey molecules) whichbind to a particular protein (bait molecule) residing on a particularbead can be identified by subjecting the individual beads to MALDI-MSand determining the unique mass and fragmentation pattern of the drugcompound(s). Thus in this embodiment the drug molecule serves on its ownas a unique mass-tag which can be distinguished from other mass tags.This is an especially useful approach since drug compounds in such alibrary are often characterized by mass spectrometry during theirsynthesis and purification. As demonstrated by the recent studyperformed using the industry-standard 384-well plate and MALDI triplequadrupole mass spectrometry [Rapid Commun Mass Spectrom. 2009 Oct. 30;23(20):3293-300. “Extending matrix-assisted laser desorption/ionizationtriple quadrupole mass spectrometry enzyme screening assays to targetswith small molecule substrates” Rathore R, Corr J J, Lebre D T, Seibel WL, Greis K D], such approach is feasible. Yet, compared to the 384-welldesign, the bead-based screening offers a significant improvement insample throughput as hundreds of thousands proteins can be assayed on asingle slide [MacBeath and Schreiber (2000) Science 289: 1760-3.].

Global Protein Expression Profiling: Expression profiling of specificproteins in a particular complex mixture such as a biological fluid ortissue or a cell culture is extremely important in current biomedicalresearch. In this application the various proteins in the sample whoseexpression level is to be measured is considered the prey moleculeswhereas specific antibodies immobilized on beads which could potentialbind to the proteins in the sample are considered the bait molecules. Inthis embodiment, different samples whose protein expression level are tobe compared are labeled with different photocleavable (PC) isotope codedmass tags. Such mass tags for example could comprise photocleavablepeptides with identical sequences but containing different isotopeslabels which thereby result in different masses for each photocleavablepolypeptide. The labeled sample is then incubated with an antibody-beadlibrary which is encoded using PC-mass tags. The relative expressionlevel of specific proteins in the sample for which there are cognateantibody-beads in the library can then be determined similar to the useof printed antibody microarrays with a fluorescent read-out. In oneembodiment, the comparison of expression level of proteins between twoor more samples is performed by (i) direct labeling of all proteins inthe sample with a PC-mass tag reactive towards, for example, primaryamine or sulfhydryl groups in the protein. The PC-mass tags have anidentical peptide sequence but variable molecular weight achieved byincorporation of isotope-labeled amino acids at specific positions; (ii)Capturing individual proteins on antibody-coated beads, which are alsocoded using a set of PC mass tags; (iii) Distributing beads intoindividual wells on a pico-plate and eluting the PC mass tags byUV-irradiation and application of MALDI matrix; and (iv) measuringrelative intensity of isotope-labeled mass tags for each of the antibodybeads.

Massively Parallel Multi-Analyte Analysis: The concentration of a largenumber of analytes in a biological or clinical sample (e.g. preymolecules) including hormones, cytokines and other bio-molecules can bedetermined similar to other bead-based multiplex assays such as theLuminex xMAP® technology based on fluorescent flow-cytometry. However,compared to Luminex which is limited to only 100 analytes, Bead-GPSallows a much larger number to be detected due to the virtuallyunlimited number of combinations of mass tags available to code the baitmolecules which are immobilized on beads.

Global Functional Screening: In one embodiment of this invention, theeffects active biomolecules (e.g. prey molecules) on a library of amass-tagged protein-bead library (bait molecules) can be determined.Such global functional screening is extremely useful in biomedicalapplications where the effects and specificity of a particularfunctional molecule such as a kinase is to be determined for a libraryof different proteins. For example, a single kinase, proteases,methylases, phosphorylases or mixtures of these molecules can becontacted with the entire bead library either before or after it isarrayed and alterations in the bait molecule residing on individualbeads along with the identity of the bait molecule determined usingMALDI-MS. This is possible because of MALDI-MS and related techniquessuch as MALDI-MS-MS to detect, at the level of individual amino acids,modifications such as phosphorylation, ubiquitination or methylation.Because each bead also contains a unique photocleavable mass tag theidentity of the bait protein residing on the bead can be uniquelydetermined. In this application it is to be noted that the preymolecules do not need to remain bound to the bait molecules but may onlytransiently interact to produce an alteration in the bait molecule. Itshould be also noted that in this application detection of theinteraction of prey molecules with the bait molecules is possiblebecause of the alteration the prey molecule produces on the baitmolecule.

Key Elements of the Present Invention

MALDI-TOF MS Imaging/Scanning of Individual Beads in an Array: MALDI-TOFMS spectra are recorded of molecules such as proteins, polypeptides,antibodies or nucleic acids attached directly or indirectly toindividual beads by directly exposing the beads to the MALDI-TOF MSlaser beam and analyzing by mass spectrometry the molecules that enterthe mass spectrometer (heretofore referred to a MALDI-TOF MS imaging ofbeads). MALDI-TOF MS bead imaging of multiple beads is accomplished byscanning over an area of the MALDI-TOF MS substrate plate in small stepsto allow sufficiently high special resolution to measure individualbeads. While examples of beads used in MALDI-TOF MS imaging given forthis invention ranged in size from 34-150 microns in diameter andconsisted of agarose beads, it is to be understood that this inventioncould also apply to larger or smaller beads of different composition.For example, smaller size beads could be analyzed using a commerciallyavailable Bruker UltraflexTreme (Bruker Daltronics) which features a 10micron focused laser for MALDI-TOF MS. In addition, various softwareprograms allow high resolution (<100 microns) to be achieved usinginstruments with only a 100 micron beam diameter, by interpolating datafrom steps smaller than 100 microns. Although Time-of-Flight (TOF) MALDImass spectrometry is the preferred method of analysis in this invention,related techniques, such as MALDI triple quadrupole (MALDI-QqQ) massspectrometry or Fourier transform MS can be used as well.

Bead-Sorted Libraries of in vitro Expressed Proteins (BS-LIVE-PRO):BS-LIVE-PRO are rapidly produced by expressing each protein separatelyin cell-free translation reactions and then binding the expressedprotein through an affinity tag to coated beads. Alternatively, singlemolecule solid-phase PCR technology (e.g. emulsion PCR) is used tocreate a Bead-Sorted Library of in vitro Expressible DNA (BS-LIVE-DNA)in single reaction and the beads then cell-free expressed into aBS-LIVE-PRO in a single reaction whereby nascent proteins are capturedonto their cognate DNA-containing bead. Advances in cell-freetranslation technology including tRNA mediated protein engineeringdeveloped by AmberGen make it possible to engineer these proteinlibraries for optimal proteomic screening [Gite, Mamaev, Olejnik andRothschild (2000) Anal Biochem 279: 218-25; Gite, Lim, Carlson, Olejnik,Zehnbauer and Rothschild (2003) Nat Biotechnol 21: 194-7; Mamaev,Olejnik, Olejnik and Rothschild (2004) Anal Biochem 326: 25-32; Olejnik,Gite, Mamaev and Rothschild (2005) Methods 36: 252-60; Gite, Lim et al.(2006) Biotechnology & Genetic Engineering Reviews 22: 151-169].

Specially Designed Pico-Well Plates Suitable for MALDI-MS andFluorescence Scanning We have developed a novel substrate consisting ofa slide with ½ million individual etched wells in the dimensions of astandard microscope slide, in order to randomly array the bead librarywith one bead per well. Both MALDI-MS and fluorescence scanning can beused to rapidly image each individual bead in the array to detectpositive biomarker hits along the identity of the protein on the beadsby using mass tags. Increased sensitivity is obtained by using a thinlayer of gold coating to neutralize charge on plate. Dimensions of thewells can be selected to control for different size beads (diameter ofwell) in addition to how far bead is exposed above slide surface (depthof well). This control is extremely to obtain optimal results such ashigh resolution MALDI MS Image scans.

Photocleavable Mass Tags: The identity of each bait molecule such as aprotein on a specific bead in the randomly arrayed library of beads aswell as positive biomarker hits based on binding of an interactingmolecule (prey molecule), such as an antibody, small molecule drugcandidate, or other biomolecule, are rapidly identified by usingAmberGen's photocleavable (PC) mass tags which are protected by over 10U.S. patents [e.g. U.S. Pat. Nos. 5,948,624; 5,986,076; 6,589,736;7,312,038; 7,339,045; 7,211,394; 6,057,096; 6,218,530; 7,057,031;7,195,874; 7,485,427; 7,547,530]. Each PC-mass tag consists of apolypeptide with a unique sequence and mass, attached through aphotocleavable linker to the bead or probe (prey), which are read byMALDI-MS. Adding a PC-mass tag to the probe (prey), such as a crudebiological sample, allows “bar-coding” of a particular sample somultiple samples can be simultaneously measured. Beads can also beencoded for specific samples by using mass tags.

Photocleavable DNA Tags: Photocleavable DNA tags provide a second methodof decoding beads and rapidly identifying biomarker hits. These tagsconsist of a unique DNA sequence linked to the bead, bait or preymolecules through a proprietary photocleavable phosphoramidite developedby AmberGen and marketed by Glen Research (Sterling, Va.).

Selective Removal of Photocleavable Mass Tags or DNA Tags from PositiveBeads: In addition to decoding of individual beads using MALDI-MS beadimaging, positive beads (beads exhibiting an interaction between theattached bait molecules and prey molecules) can be decoded by selectivephotocleavage of the mass tags. For example, a conventional fluorescencescanner can be modified to include a laser with excitation wavelengthand intensity sufficient to photocleave mass tags or DNA tags from thebeads when the fluorescent scan detects a positive hit (interaction).For example, such a device would allow collection of all mass tags orDNA tags from positive beads in a bead array such that the tags couldthen be analyzed with an appropriate reading device. In the case of masstags, the entire set of selectively removed mass tags could be readusing a conventional (non-scanning) mass spectrometer in a singlemeasurement. Similarly, the DNA tags could be read using an RT-PCRdevice containing suitable probes for each DNA tag or by usingmassively-parallel sequencing of individual DNA tags. The advantage ofthis approach is that a single measurement allows all positive beads inbead array to be identified.

Fluorescence and MALDI MS Image Synchronization for Bead Selection: Animportant feature of one embodiment of this invention is the ability tosynchronize the fluorescent scan with the MALDI-MS decoding therebyreducing the number of beads which need to be scanned by MALDI-MS . Inorder to improve accuracy, synchronization beads consisting of afluorescent label and unique mass tag can be added in the random beadarray. Such synchronization beads allow more accurate registration ofthe fluorescence and MALDI images. In addition to pico-wells formed fromfiber optic bundles, a photolithographic method of well fabrication canbe utilized to increase accuracy of the position of each well.

Physical Bead Selection Prior to MALDI-MS Analysis or Imaging: Analternative (or complement) to direct selection of positive hits (beads)by fluorescence imaging prior to MALDI-MS decoding is the use ofphysical methods to separate positive beads from negative beads. Onesuch approach is the use of fluorescence activated cell-sorting (FACS)(see Experimental Example 11). A second method is use of a magnetic beadsorting techniques.

Although selection either by imaging or physical separation of positivehits (beads) prior to MALDI-MS decoding is the preferred method, itshould be noted that it is not required. As demonstrated, mass tags canbe used alone for both bead identification and autoantibody readout (seeExperimental Examples). In this case, since hits are not pre-imaged byfluorescence, the entire library is imaged by MALDI-MS in the pico-wellplates. Importantly, the newer generation of faster scanning MALDI-MSinstruments can do this in a relatively short amount of time.

Mass Tags for Bead Decoding

Basic Concept: Once beads have been sorted or selected for positive“hits” on the basis of fluorescence scanning as described above, thenext step in one embodiment of this invention is decoding the beans. Wehave developed a new method of decoding based on the use ofphotocleavable mass tags. In one embodiment of this invention, the masstags evaluated are modified polypeptides whose sequence has been chosenso that its mass is unique (i.e. differs from every other mass tag usedin the library). In a second embodiment, the mass tags are isotopicallylabeled polypeptides with the same sequence but different masses. In athird embodiment, the mass tags consist of a different polymer than apolypeptide such as an oligonucleotide. It is to be understood that inthis invention there are a variety of molecules which would serve asmass tags and it is not limited to one class of molecule or polymer.

In principle, a relatively small peptide (e.g. an octamer, N=8) canprovide sufficient number of sequences to provide sufficient uniquemasses to satisfy even a large library of 100,0000 different “bait”species (20^(N)≈25×10⁹). In practice, the number of viable sequencesdepends on the mass resolution of the MALDI-MS instrument which is oftenbetter than 0.1 daltons in the mass range measured. In addition, anydegeneracy in the molecular weight of the mass tags can be decoded usingthe ability of MALDI-MS to sequence small peptides (<5,000 MW).Additional “fine-tuning” of masses can be accomplished by modificationof the mass tag such as the addition of fluorescent labels.

As shown in FIG. S04, multiple mass tags can be deployed on each bead todetermine the identity of the attached protein (red), the sample beingscreened in cases where multiple samples are scanned (sometimes referredto as bar-coding) (purple) and the presence of an interacting probe orprey (e.g. antibody) indicating a positive hit or biomarker (green).Since, as described above, beads can be pre-selected by fluorescencescanning on the pico-well slide, this last mass tag serves to reducefalse-positives ensuring higher accuracy for biomarker selection.

Photocleavable Linkers

In some Experimental Examples shown in this patent, which used mass tagsattached to beads for identification, the mass tags were not directlycovalently linked to the bead surface but instead bound through anantibody-polypeptide interaction (e.g. Example 3). However, this isnon-ideal since stringent wash steps could result in partial removal ofthe tags. One solution to this problem is to use covalently attachedmass tags which are photo-released upon exposure to the proper light.Alternatively, a near-covalent strength linkage between (strept)avidinand biotin (K_(d)=10⁻¹⁵) can be used in conjunction with aphotocleavable linker.

AmberGen has developed a novel class of photocleavable linkers(PC-Linkers) useful in a variety of applications such as photocleavageassisted molecular purification, tRNA-mediated protein engineering,photo-activation of compounds, bio-molecules and viruses as well asphotocleavable mass-tagging for multiplexed assays [Olejnik,Krzymanska-Olejnik and Rothschild (1996) Nucleic Acids Res 24: 361-6;Olejnik, Krzymanska-Olejnik and Rothschild (1998) Nucleic Acids Res 26:3572-6; Olejnik, Ludemann et al. (1999) Nucleic Acids Res 27: 4626-311.In the case of mass-tagging of the proteomic bead-library, a shortpeptide with 7 or 8 amino acids is linked to the beads via aphotocleavable linker. Note that previous experiments have demonstratedthat AmberGen's PC-Linker is rapidly photocleaved with 95% efficiency isless than 10 minutes using a low-intensity commercial black-light[Olejnik, Ludemann et al. (1999) Nucleic Acids Res 27: 4626-31].

In one embodiment of the invention photocleavable (PC)-mass-tags forprotein identification are attached to beads in one of two ways asillustrated in FIG. S05 and detailed below:

Ultra-High Affinity Biotin-(Strept)Avidin: Peptide mass tags modified atthe N-terminus, for example, with AmberGen's photocleavable biotin areattached to (strept)avidin coated beads. This mass-tagging method hasalready been demonstrated in the Experimental Examples (see Example 5).

Direct Covalent: Peptide mass tags bearing an N-terminal photocleavableprimary amine moiety can be chemically attached to beads. In the examplein FIG. S05, NHS-activated (primary amine-reactive) beads are used forthis procedure. This is highly analogous to AmberGen's phosphoramiditetechnology distributed through Glen Research (Sterling, Va.) forintroducing a photocleavable primary amine at the 5′ end of DNA[Olejnik, Krzymanska-Olejnik et al. (1998) Nucleic Acids Res 26:3572-6]. Here, peptide mass tags lacking lysines (reactive primary amineon side chain), or where lysines are blocked on the ε-amine, will beused to avoid non-cleavable attachment to the amine-reactive beads.

In one embodiment for the production of PC-mass tag libraries, a libraryof peptides pre-screened by mass spectrometry can be commerciallysynthesized by available vendors such as Mimotopes (Austria), Peptide2.0 Inc. (Chantilly, Va.) or GenScript Inc. (Piscataway, N.J.) and usedto create the mass tags which will be photocleavably linked to thebeads. High throughput peptide synthesis services are available fromthese vendors (e.g. soluble peptide arrays in 96-well plates) andpeptides can be purchased with full HPLC and mass spectrometry qualitycontrols. Conventional solid-phase chemical peptide synthesis begins atthe C-terminus and ends at the N-terminus. The growing peptide istethered to the solid-phase synthesis resin via its C-terminal carboxylgroup, exposing its N-terminal amine (after deprotection) and allowingsequential attachment of another N-terminal blocked amino acid precursor(again followed by deprotection). Thus, the attachment of N-terminalmodified PC-Biotin or PC-amine (amine protected) amino acid precursorsat the final cycle of synthesis would be a relatively strait forwardprocess.

We have estimated that due to the high analytical sensitivity of massspectrometry (attomoles), even adding 10 fmoles per bead of mass tags(10-mer), the aforementioned peptides with N-terminal PC-Linkermodification and all quality control data will add only pennies (¢10) tothe cost of an entire proteome-bead library (e.g. at 500,000beads/library). Note that data in the Experimental Examples has alreadyshown that strong MALDI-TOF MS mass-imaging signal can be achieved when5 fmoles/bead is added (5 fmoles maximum assuming 100% binding; seeExample 5).

In addition to PC-mass tags attached to the beads for identificationpurposes, the present invention can utilize PC-mass tags attached to theprobes used to query the bead library. In the example of using proteomebead-libraries for autoantigen discovery, the PC-mass tag is attached tothe anti-[human IgG] secondary antibody probe used to detect the boundserum autoantibody. In this case, only one species of unique mass tag isrequired. This has already been experimentally demonstrated inExperimental Example 7. In one embodiment of the invention, customreagents can be synthesized to allow direct covalent labeling of probes(e.g. antibodies) with PC-mass tags (FIG. S06).

MALDI-MS for Mass-Imaging of Individual Beads

An important feature of one embodiment of this invention is the abilityof MALDI-MS to rapidly scan/image individual beads. In particular, wehave demonstrated (see Examples) that MALDI-MS is capable of rapidlydecoding PC-mass tags, including for example in conjunction withread-out antibodies (probes) to detect positive autoantibodyinteractions with protein autoantigens (bait) on beads. This capabilityto quantitatively measure, using mass spectrometry, molecules fromindividual beads offers many advantages not limited to just mass tagdecoding, but also for direct identification of proteins and otherbio-molecules residing on the bead surface or indirectly attached to thebead.

Instrumentation and Software

As an example of the ability of MALDI-MS to image individual beads,measurements were performed using an ABI 4800 Plus MALDI-TOF-TOF massspectrometer (see Examples), although such measurements are not limitedto this particular model of MALDI-TOF instrument. Typically, scanning isdone using the ABI 4800 software in the positive ion reflector mode withinternal calibration using 50-200 laser pulses per sample spot, whichresults in measurement times of ˜0.25-1 s per bead.

In general, mass spectrometry and MALDI-MS in particular have proven tobe highly amenable to high throughput applications in both clinical andbasic research settings. For example, Sequenom Inc. (San Diego, Calif.)has established MALDI-MS as an effective technique in the field ofgenotype profiling, and is providing diagnostic products in this area.As a second example of automation of mass spectrometry in clinicaldiagnostics, the Pediatrix Medical Group (Sunrise, Fla.), the largestprovider in the US for neonatal blood tests, uses tandem array massspectrometry to detect metabolic disorders and has screened over 2million babies using this method.

In the case of this invention, many improvements are envisioned whichcan facilitate automation and high throughput biomarker discovery. Forexample, multiplexing can be achieved at several stages including duringthe preparation of the bead library and in bar-coding multiple sample.Importantly, the use of a highly automated mass spectrometer such as theABI 4800 Plus MALDI-TOF-TOF MS or the more advanced ABI 5800 will alsofacilitate high throughput analysis at the MALDI-MS bead scanning stage.For example, this system uses advanced software designed for automatedscanning of a two-dimensional area, data collection and spectralprocessing. As an example, we already demonstrated the ability toautomatically scan approximately 10,000 beads in the pico-well MALDIplate in one hour (see Examples). The use of the ABI 5800 should reducethis time to 1/10 (6 minutes). Furthermore, using one of thecommercially available plate-loading robots will allow the use of theinstrument in operator-free mode, 24-hours a day. As an example ofautomation levels achievable with MALDI-MS, Sequenom, Inc. hasintroduced a MALDI-based system for SNP analysis which is capable ofanalyzing 100,000 genotypes per day.

Finally, the MALDI-MS imaging of individual beads described in thisinvention requires software to analyze data and to identify mass tags onindividual beads. There are a variety of software packages availableboth commercially and in the public domain for this purpose. As anexample, we performed image acquisition and analysis using public domainsoftware 4000 Series and BioMap software respectively(www.maldi-msi.org). BioMap is a powerful biomedical image analysissoftware package supporting various data types generated by optical,PET, CT and mass spectrometry based imaging. The BioMap platform allowsvisualization and storage of large volumes of data includingexperiment-specific information such as scan ID, experimental protocoland sample history. It is also a flexible tool that can be easilymodified to accommodate a specific requirement. It is contemplated thatas part of this invention, improved software for imaging of individualbeads and mass tags can be made that is designed for a MALDI-MSbead-imaging workflow, such as automated co-registration of fluorescentand MALDI-MS scan images and identification of positive “hits” based onthe detection of PC-mass tags.

Mass Tag Decoding

In general, a requirement of mass tag decoding is that each mass tagpeak must correspond to the correct molecular weight predicted by themass tag molecular structure, such as for example a given polypeptidesequence plus any modifications or isotope labeling, within theresolution of the spectrometer in the specific mass range (˜0.1 Da in600-4,000 Da range).

It is highly desirable that each mass tag peak must have asignal-to-noise ratio of at least 50:1, although lower signal-to-noiseis sufficient for some applications. For comparison, the signal-to-noiseratio of single prototype mass tags attached to beads incorporated intoarrays as described in the Experimental Examples are routinely >50:1using a set of standard mass spectral parameters. Note that thesignal-to-noise ratio in all experiments is determined using the ABI4800software, which measures the integrated target peak intensity and ratiosthis to the integrated intensity of a nearby background region whichexhibits no detectable peaks.

Importantly, spectral resolution and mass accuracy of the ABI 4800 PlusMALDI-TOF-TOF analyzer is sufficient to unambiguously identify peptidesseparated by as little as 0.1 Da. However, one potential problem is theappearance of several peaks for each peptide in the mass spectra, whichare separated by 1 Da (the “isotope envelope”), due to the presence ofsmall amounts of mass-shifted C13 and N15 atoms in the protein sequence.In the case of two mass tags separated by only a few Daltons, thespectral overlap may affect the tag identification. This will beaddressed by using, in real-time, a spectral processing routine calledpeak de-isotoping. The routine, which is built-in into the ABI 4800 dataacquisition software, replaces multiple peaks in the isotope envelopewith a single mono-isotopic peak (corresponding to the sequencecontaining only C12 and N14 atoms).

In Situ Mass-Fingerprinting of Proteome Bead-Arrays

Rather than the addition of exogenous mass tags, or any other tags, itis possible to utilize the bead-bound bait molecules (e.g. recombinantproteins) themselves as identification codes. Analogous to massspectrometry based on mass-fingerprinting used in classical proteomics,proteins are digested with protease (e.g. trypsin) and the resultantpeptide “fingerprint” used for protein identification. If necessary, thepeptides are further fragmented in the TOF/TOF tandem mass spectrometerand sequenced using the standard capabilities of today's instruments.Experimental Examples 8 and 9 demonstrate this capability in MALDI-MSbead imaging mode.

Photocleavable DNA Tags

In addition to peptide based decoding of a bead library, we havedeveloped an alternative method of coding individual beads based on theuse of photocleavable (PC)-DNA tags. Such tags are also based onproprietary photocleavage technology developed by AmberGen [U.S. Pat.Nos. 5,948,624; 5,986,076; 6,589,736; 7,312,038; 7,339,045; 7,211,394;6,057,096; 6,218,530; 7,057,031; 7,195,874; 7,485,427; 7,547,530] whichoffers convenient synthesis of DNA molecules with a 5′-modificationconsisting of a photocleavable linker such asPC-aminotag-phosphoramidites commercially distributed by Glen ResearchInc. These tags can be directly linked to the activated beads similar tothe method used for PC-mass tags and released upon exposure to near-UVlight (see Experimental Examples). Once removed from individual positivebeads, these PC-DNA tags can be rapidly decoded and quantified in bulkusing a massively parallel PCR or sequencing platform. One embodiment ofthis is shown in Experimental Example 10. In addition, MALDI-MS beadimaging of PC-DNA tags from beads or particles is also possible.

Importantly, photocleavage from individual beads and collection of DNAtags (or mass tags) from positive beads can be easily accomplishedalmost simultaneous with fluorescent scanning by using a modifiedfluorescence microarray scanner. In particular, a laser normally usedfor scanning the image can be replaced with a laser capable ofphotocleavage of DNA tags from individual beads such as a pulsed Nd—Yaglaser with 355 nm output which are widely commercially available at lowcost. It has been demonstrated by us that such lasers canphotocleave >90% of the tags on a bead sample in less than a fewseconds. Since commercial fluorescent scanners operating with multiplewavelengths and different lasers are designed to perform sequentialscans maintaining image registration, software image identification ofpositive beads would allow the Nd—Yag laser to be switched on to exposeonly positive beads during a sequential registered scan. Scan resolutionis normally 3-5 microns, allowing high precision for Nd—Yag laser beamto photocleave DNA tags from 34 micceon beads located in the pico-wellplate (beads used extensively in Experimental Examples). Alternatively,a scanner using a CCD imager along with a photocleaving laser can bereadily used to selectively remove DNA tags from specific beadsidentified as positive in the fluorescent scan. Photocleaved DNA tagscan be collected in a thin fluidic chamber overlaying the array forsubsequent decoding. Importantly, selection of the positive hits issimplified for this approach since the imaging and photo-release aredone simultaneously in the same instrument.

DETAILED DESCRIPTION OF THE INVENTION

Example of Approach: A simplified flow diagram for one embodiment of thepresent invent designed for discovery of autoimmune biomarkers is shownin FIG. S07. It consists of several steps briefly described below and inmore detail in the following sections.

1. Sample: For the discovery process, the sample consists of blood seraobtained from patients with a known autoimmune disease or no knowndisease (control). Auto antibodies that are typically present in suchautoimmune disease patients constitute the prey molecules in thisinvention

2. Probe Proteomic Bead-Library with Sample: The sera are mixed with aproteome bead library prepared using recombinant or cell-free proteinexpression methods. The full library will encompass 15,000 unique humanproteins and 20 bead replicates for each protein in the library (total300,000 beads). These proteins constitute the bait molecules in thisinvention and are also potential autoantigens or biomarkers that can beutilized in diagnostic tests for autoantigen diseases. In someapplications, after mixing the proteomic bead-library with sample, thebeads are randomly arrayed into a pico-well plate suitable for bothfluorescent and MALDI-MS imaging.

3. Selection: Positive beads that interacted with autoantibodies presentin the sera are selected for decoding. A variety of methods are providedfor selection. In one preferred method, beads are first scanned forfluorescence which originates from fluorophores that are bound directlyor indirectly to the autoimmune antibodies through binding agents. Inone embodiment, the fluorescence label is bound directly to a secondaryantibody which is specific for the class of antibodies in the sera (e.g.human IgG). In this case, the secondary antibody serves as the bindingagent. In all cases, autoantibodies which bind to beads will result influorescence emitted from the beads where prey molecules interact thatis detected by conventional fluorescent scanners. In a secondembodiment, the beads which are positive are identified usingflow-cytometry often and physical separated from other beads (FACS). Ina third method, the beads which are positive are physically separatedusing magnetic beads.

In another embodiment, the positive beads are selected using MALDI-MSbead imaging wherein the mass-tag is attached to the prey molecule,either directly or indirectly through a binding agent. For example, theprey molecule could be an autoantibody directed at a specificautoantigen on an individual bead. In some cases the mass tag isattached via a binding agent such as a secondary antibody. In thesecases, positive hits are identified by detection of the specific masstag associated with the prey molecule.

It is also to be understood that in the present invention different masstags can be attached directed or indirectly to different prey moleculesin order to distinguish which prey molecules interact with which baitmolecules. For example, in the case of studying protein-proteininteractions, a library of proteins can be coded by attaching differentmass tags to each type of protein in the library. Once a protein in thelibrary (prey molecule) interacts with a specific bait molecule on aspecific bead, its identity can be determined because of its attachmentto a specific mass tag which is different from other mass tags used tocode prey molecules and specific bead types.

In another example, different mass tags are used to code autoantibodiesoriginating from different samples. In this embodiment, each samplewhich may containing autoantibodies are mixed with a suspension of beadsand then the autoantibodies allowed to interact with the bait moleculesresiding on beads. Mass tags specific for each sample are then attachedto autoantibodies (prey molecules) through a binding agent which in thiscase is a secondary antibody. The various bead suspensions exposedindividually to different samples are then mixed together and an beadarray formed which is measured using MALDI-MS-Imaging. The beads whichhave bound prey molecules can then be identified along with the samplewhich the prey molecules originated since each prey molecule(autoantibody) has a unique mass tag.

4. Decoding: The beads selected in the previous step as positive aredecoded to determine the identity of the protein residing on the bead.This is accomplished by measuring the mass of the photocleavable masstag present on the selected bead by MALDI-MS. A variety of other methodsare also described in this invention to determine the identity ofphotocleavable tag including RT-PCR and DNA hybridization.

5. Validation: Using a new patient cohort, each individual biomarkerdetermined in steps 1-4 is clinically validated by testing for itsdiagnostic sensitivity and specificity using an independent method suchas two-epitope solid-phase T²-ELISA™ assay based on cell-free proteinsynthesis technology.

Bead Library Construction

Several methods can be used to construct the proteomic-bead library. Onemethod termed Parallel Preparation, consists of cell-free expression ina separate reaction of each protein in the library from a speciallydesigned plasmid containing the target gene. This is followed by bindingof the expressed protein to beads and pooling of all beads in abead-library. A second method described in US Patent Application Nos.20090264298, 20090270278, 20090286286, 20100062451 and 20100075374,termed here as the Batch Preparation method, involves only two reactionsto create the entire library and relies on advanced methods utilizingsingle-molecule solid-phase emulsion PCR and self-assembling cell-freeexpression.

We described below several examples of steps used in bead libraryconstruction in the present invention:

Plasmid Construction: As illustrated in FIG. S08, we have developed afacile approach for constructing the plasmids used in the cell-freeprotein translation reactions which is based on the commerciallyavailable 12,000-member Open Reading Frame (ORF) template library(ORFeome) [Rual, Hirozane-Kishikawa et al. (2004) Genome Res 14:2128-35] derived from NIH backed Mammalian Gene Collection. This librarycan be transferred into a custom in-house developed destination vector(pVSV-DEST vector) using the high throughout Gateway™ recombinationmethod (Invitrogen). The Gateway™ system allows flexibility byfacilitating recombination into a variety of expressible acceptor(destination) vectors. Our pVSV-DEST acceptor vector affords excellentexpression in a variety of cell-free reaction mixtures including E.coli, rabbit reticulocyte and wheat germ lysates. Gateway™ compatibleacceptor vectors for insect cell lysate expression systems have alsobeen prepared by AmberGen. In addition to the Open Reading Frame (ORF)inserts, nucleic acid sequences coding for common epitope tags at the N-and C-terminal end of the nascent protein are included (a key feature ofthe ORFeome is that the stop codon is absent, allowing for N-Tagging).The Gateway™ process is rapid, facile and efficient and avoids tedioussub-cloning.

Cell-free Protein Transcription & Translation: Customized plasmidconstructs as described above will be used to express the requiredprotein for the library in a cell-free (in vitro) expression system.Until recently, relatively high costs and low yields have discouragedthe use of cell-free protein expression systems in the field ofproteomics. However, recent dramatic improvements in this field holdgreat promise for solving many of the problems that have previouslylimited the use of this approach. Advantages and improvements include:On-Demand Expression: Express specific proteins, on-demand, typically in<1 hr, even in eukaryotic (e.g. mammalian) systems using a single facilereaction (e.g. Promega's batch mode rabbit reticulocyte or insect cellcoupled transcription/translation system). High Yield: New “continuousexchange” cell-free (CECF) expression systems capable of ˜1 mg/ml,yields (e.g. Roche's Wheat Germ CECF now sold by 5 Prime, Inc.). Inthese systems, the expression reaction is separated from a feedingbuffer by a semi-permeable membrane. The feeding buffer provides acontinuous supply of small molecule precursors while absorbing (bydiffusion) inhibitory byproducts. The devices (FIG. S09) fit into a96-well microtiter plate frame for automation. Protein Compatibility:Often proteins cannot be expressed in cellular systems due tocytotoxicity or interference with host cell physiology [Hienrich, Lubitzet al. (1982) Mol Gen Genet 185: 493-7; Goff and Goldberg (1987) J BiolChem 262: 4508-15; Nakano and Yamane (1998) Biotechnol Adv 16: 367-84;He and Taussig (2001) Nucleic Acids Res 29: E73-3; Endo and Sawasaki(2003) Biotechnol Adv 21: 695-713]. Membrane Proteins: The ability toexpress membrane proteins in nanolipoparticles [Cappuccio, Blanchette etal. (2008) Mol Cell Proteomics 7: 2246-53; Katzen, Fletcher et al.(2008) J Proteome Res 7: 3535-42; Cappuccio, Hinz et al. (2009) MethodsMol Biol 498: 273-96], small discoidal membranes mimicking the nativemembrane protein environment, using commercial kits such as theInvitrogen MembraneMAX™ cell-free protein expression kits [Cappuccio,Blanchette et al. (2008) Mol Cell Proteomics 7: 2246-53; Katzen,Fletcher et al. (2008) J Proteome Res 7: 3535-42; Cappuccio, Hinz et al.(2009) Methods Mol Biol 498: 273-96].

Since the proteomic-library will consist of a wide-variety of differentproteins it is essential that cell-free reactions are designed to becompatible with expression of these different types of proteins.However, there is abundant evidence that such a diversity of proteinscan be universally expressed if the proper system is chosen. Forexample, recent studies reported in Nature Methods [Goshima, Kawamura etal. (2008) Nat Methods 5: 1011-7] using wheat germ expression system hasbeen shown to be capable of expressing at least 13,346 proteins from theproteome. The expression system was a variant of the aforementioned highyield CECF system, except that as an alternative to the 2-chambereddevices, the so-called “bilayer” method was used to overlay the feedingbuffer directly onto the expression reaction [Sawasaki, Hasegawa,Tsuchimochi, Kamura, Ogasawara, Kuroita and Endo (2002) FEBS Lett 514:102-5]. Recently, synthetic nanolipoparticles (NLP) [Cappuccio,Blanchette et al. (2008) Mol Cell Proteomics 7: 2246-53; Katzen,Fletcher et al. (2008) J Proteome Res 7: 3535-42; Cappuccio, Hinz et al.(2009) Methods Mol Biol 498: 273-96], small discoidal membranesmimicking the native membrane protein environment, have been used forcell-free expression of properly folded and functional membraneproteins.

In one example of the prototype library construction used in thisinvention, we have used the Promega rabbit reticulocyte cell-freeexpression system, known for its ability to produce functional, properlyfolded and even post-translationally modified proteins, includingmulti-pass membrane proteins, especially considering it is a mammaliansystem, and as a cell lysate, native chaperones are not removed [Gibbs,Zouzias and Freedberg (1985) Biochim Biophys Acta 824: 247-55; Hirose,Kim, Miyazaki, Park and Murakami (1985) J Biol Chem 260: 16400-5;Vorburger, Kitten and Nigg (1989) Embo 18: 4007-13; Pensiero, Dveksler,Cardellichio, Jiang, Elia, Dieffenbach and Holmes (1992) J Virol 66:4028-39; Middleton and Bulleid (1993) Biochem J 296 (Pt 2): 511-7;Popov, Tam, Li and Reithmeier (1997) J Biol Chem 272: 18325-32; Lyfordand Rosenberg (1999) J Biol Chem 274: 25675-81]. Examples of two highyield systems which can be used in conjunction with library constructionare: i) insect cell coupled transcription/translation systems (Promega)and ii) wheat-germ continuous flow system (Roche; now sold by 5 PrimeInc.). FIG. S10 shows that the 100-fold expected yield improvement ofthe Wheat Germ CECF system over the rabbit reticulocyte is in factachieved. In this example, a known autoantigen, snRNP C, was cell-freeexpressed in both systems and analyzed by the AmberGen's aforementionedT²-ELISA™ assay. To avoid saturation of the capture antibody on theELISA plate, the expression reaction was serial diluted prior to inputinto the assay. At the lowest expression input, the wheat germ system ismeasured at 94-fold better yield than the rabbit reticulocyte, in linewith the manufacturer reported yields of the 2 systems (600 and 7 ng/μLrespectively).

One-step Capture of Protein on Bead: As illustrated in FIG. S11, in oneembodiment of the present invention each expressed protein in thelibrary is purified and attached to the bead through a simple one-stepprocess. This is accomplished through the common HSV-epitope (C-Tag)incorporated into each protein which binds to an anti-HSV antibodypresent on the 34-micron agarose beads. We have measured a 75% captureefficiency from cell-free expression lysates with this antibody system[Lim and Rothschild (2008) Anal Biochem 383: 103-115]. It should also benoted that we have shown this attachment method to be extremely stable.For instance, after overnight incubation of a mixed population ofprotein-beads at room temperature with vigorous shaking, no significantdrop in signal and no bead-to-bead cross-contamination was observed.

As an alternative to the above approach, tRNA mediated engineeringtechnology such as described in various patents issued to AmberGen Inc.[e.g. U.S. Pat. Nos. 5,643,722; 5,922,858; 6,210,941; 6,303,337;6,306,628; 6,344,320; 6,358,689; 6,566,070; 6,596,481; 6,875,592;6,949,341; 7,169,558; 7,252,932; 7,288,372 and 7,524,941] can be used toco-translationally incorporate biotin labels for attachment to(strept)avidin beads [Lim and Rothschild (2008) Anal Biochem 383:103-115], thereby exploiting the unparalleled affinity of thisinteraction (Kd=10⁻¹⁵, roughly 6-orders of magnitude better than anaverage antibody).

An important feature of the present invention when agarose beads areused is that it exploits the extremely high protein binding capacity ofthe porous 6% cross-linked 3-dimensional matrix of the 34 micron agarosebeads. According to the manufacturer's specifications (GE LifeSciences), the chemical surface activation (primary amine-reactive NHSgroups) of the uncoated agarose beads is 10 μmoles/mL of packed beadvolume, which would correspond to 50,000 pg/bead of antibody attachmentcapacity. However, in practice, due to steric factors, the much largerantibody cannot be loaded to such levels. The typical antibody bindingcapacity of such beads is ˜10 mg/mL of beads, or ˜300 pg or 2fmoles/bead (1 mL 30 million beads). This still compares favorably toconventional 2-dimensional planar proteome microarrays such asInvitrogen's ProtoArrays®, with published densities of 1 pg/spot maximum(100 micron spots) [Zhu, Bilgin et al. (2001) Science 293: 2101-5].Furthermore, this should easily facilitate our targeted intended loadingof 10 pg (0.2 fmoles) per bead of expressed protein on average, inaddition to the loading of the peptide mass-tags. Note that because anantibody is larger (150 kDa) than a typical protein (calculations on 50kDa), 30 pg/bead (0.2 fmoles) of antibody is targeted.

Another important feature of the bead-based capture is the ability toeasily normalize for protein-protein variances in expression yield (seeFIG. S12).

Automation of Bead-Library Fabrication: An important feature of thebead-library fabrication is the ability to fully automate the process.This ability stems from relatively few steps involved in the overallpreparation of the protein-beads. In essence, because of the cell-freeexpression and in situ protein capture, the fabrication of the beadlibrary is only a series of steps involving mixing of reagents andwashing beads. In particular, each step can be carried out in parallelon standard 96-well plates using our in-house robotic plate handlingsystems. For example, transfer of the genes from the ORFeome library tothe pVSV-DEST expression vectors essentially involves a singlerecombination step using the Gateway™ recombination system. In fact,high throughput automated Gateway™ recombination has been done with 93%efficiency without the need for bacterial plating and/or colony pickingat any stage [Janney, Roby, Getbehead, Bell, Daniels and Chesnut 2009;Aguiar, LaBaer et al. (2004) Genome Res 14: 2076-82]. Similarly,expression of the protein in a high yield coupledtranslation/transcription reaction requires just one step which involvesmixing reaction components and vector in a tube followed by incubationat a controlled temperature. The purification of the protein from thereaction system and binding to beads also involves a single-stepprocess. Since our library encompasses ˜15,000 proteins, this willrequire methods that involve robotically processing approximately150×96-well plates for each of the 5-steps (note we anticipate that theORFeome will expand to this number from its currently available 12,000clones). This number of plates can be easily accommodated using industrystandard multi-plate stacking devices which are compatible with ourrobotic systems.

Alternative Batch Library Production with Advanced Single-MoleculeTechnologies: As an alternative to the Parallel Preparation method offabricating the proteome bead-libraries in the present invention, analternative method described in US Patent Application Nos. 20090264298,20090270278, 20090286286, 20100062451 and 20100075374 is potentiallymore efficient and cost effective. This fully multiplexed BatchPreparation method uses a single-tube reaction to first produce anentire bead sorted library of in vitro expressible DNA (BS-LIVE-DNA).For this, universal PCR primer beads (vector targeted) are emulsified inoil with the expressible ORFeome library as template such that singlebeads are compartmentalized in aqueous droplets (millions per reaction)containing single copies of template. The emulsion PCR reaction thusclones and amplifies single DNA molecules onto beads, and is normallyused for genetic assays [Dressman, Yan, Traverso, Kinzler and Vogelstein(2003) Proc Natl Acad Sci USA 100: 8817-22]. After loading the commoncapture antibody onto the resultant BS-LIVE-DNA beads (via abiotin-(strept)avidin bridge), the bead population is subjected to asingle-tube self-assembling cell-free expression reaction [Nord, Uhlenand Nygren (2003) J Biotechnol 106: 1-13; Ramachandran, Hainsworth etal. (2004) Science 305: 86-90] to convert BS-LIVE-DNA to a bead sortedlibrary of in vitro expressed proteins (BS-LIVE-PRO). In this reaction,proteins are simultaneously translated and captured back onto the parentDNA beads from which they were made, by way of the capture antibodydirected against a common epitope tag in each protein. Although notrequired per se, methods to reduce mRNA/protein escape from its parentDNA encoded bead can be employed, such as expression in an emulsion orin the pico-well plates to reduce diffusion.

MALDI-MS Measurements of Individual Bead in an Array

An important feature of this invention is the demonstrated ability ofMALDI-MS to rapidly measure a variety of molecules residing onindividual beads as part of an array of beads. In one preferredembodiment of this invention this ability allows decoding of individualbeads that have been identified as displaying positive interactionsbetween bait molecules residing on bead and prey molecules whichinteract with said bait molecules. In several example described in thisinvention, we have demonstrated that MALDI-MS is capable of rapidlydecode PC-Mass-Tags on beads, including their utilization to detectionthe interaction of autoantibodies with autoantigens residing onindividual beads. In general this ability to measure molecularinteractions on individual beads using MALDI-MS, offers many advantagesnot limited to just mass-tag decoding but also for direct identificationof proteins and other biomolecules residing on the bead surface orindirectly attached to the bead as described below.

Several examples of the ability of MALDI-MS to image individual beadsthat were performed using an ABI 4800 Plus MALDI-TOF-TOF massspectrometer (see Examples below) although such measurements are notlimited to this particular model of MALDI-TOF instrument. Typically,scanning is done using the ABI 4800 software in the positive ionreflector mode with internal calibration using 50-200 laser pulses persample spot, which results in measurement times of ˜0.25-1 s per bead.Image acquisition and analysis is performed using public domain software(www.maldi-msi.org; 4000 Series and BioMap software respectively).

Fluorescent Selection of Positive Beads

Fluorescent Scanning of Beads in Pico-well Plate: In one embodiment ofthe present invent designed for biomarker discovery involving autoimmunedisease , hits corresponding to proteins that interact with autoimmuneantibodies in the sample serum (i.e. putative biomarkers) are selectedby detection of fluorescent spots on a conventional proteomicmicroarray. In contrast, the present invention relies on identificationof positive beads. This can be accomplished in several ways such as butnot limited to fluorescent scanning of the beads residing in a pico-wellplates after exposure to the test sera. The positive beads identifiedcan then be decoded using MALDI-MS bead scanning and positive hitsfurther confirmed by detection of mass-tags attached to the read-outantibody.

Fluorescent Labeling of the Probe (Prey) Molecule: There are a multitudeof fluorescent based methods commonly used to identify the interactionof a bait molecule with probe (prey) molecule. One common method isbased on using a fluorescently-labeled antibody directed against theprobe molecule which is applied either before or after the bait-preyinteraction occurs. In the case of autoimmune antibodies residing inpatient sera for example which are themselves the prey molecules, afluorescently labeled secondary antibody is used which is directedagainst the autoimmune antibody. Alternatively, a fluorescently labeledstreptavidin molecule can be used directed at a biotinylated antibodywhich is in turn directed against the probe molecule such as anautoimmune antibody. In all of these cases the antibody serves as abinding agent coupling the prey molecule with a fluorescent molecule.The binding agent might also comprise a group residing on the preymolecule which facilitates a covalent linkage with the fluorescent labelsuch as through an activated or reactive chemical group. For example,the epsilon amino group of lysines residing on proteins can bechemically labelled with appropriate fluorophores which are commerciallyavailable. It is to be understood that labeling of the probe moleculesis not limited to a single fluorescent molecule. In some embodiments itis advantageous to use different fluorescent labels for different preymolecules that might be coupled to the prey molecule through differentbinding agents such as antibodies or reactive chemical groups. It isalso to be understood that probe molecules can also comprise a codingagent such as a photocleavable mass tag.

As listed below, there are several key advantages to this approach overconventional microarrays including:

More Reliable Hit-Identification due to Increased Replicates: Theability to scan in a few minutes a large bead library of beads such as½-million beads arrayed on the pico-well substrate provides morereliable hit-identification than conventional microarrays. In comparisonto the typical <20,000 spots with 2-replicates used on the conventionalproteomic microarrays, the current invention can scan significantly morereplicates (˜20) providing better statistics for identifying weakprotein-antibody interactions.

False-Positive Rejection: Since in one embodiment of the invention theprobe antibody is doubly labeled with not only a fluorescent label but aphotocleavable mass-tag designed for MALDI-MS bead imaging read-out,this assures that any false positive beads selected during the sort stepwill be rejected during decoding. See FIG. 7 (Experimental Examples) forone embodiment of this redundant design.

Multi-Dimensional Hit Identification: The use of multiple fluorescentlabels with different wavelengths of excitation provides more robustability to separate different positive hits. For example, the probeantibody can be coded with different fluorescent dyes in order todifferentially label different samples scanned. Such a capability is notpossible using conventional microarrays since the sera is applieddirectly to chip instead of to beads in separate reaction vessels priorto application to pico-well slide.

Non-Dry Conditions: Conventional proteome microarray fabricationinvolves several steps which subject the proteins to non-physiologicalconditions. For example, printing followed by drying can lead to proteindenaturation. Microarrays are also stored and shipped dry for longperiods of time which can lead to further protein alterations. Inaddition, antibody interactions occur on two-dimensional protein spotsprinted on an array surface which could alter the ability of theantibody to freely interact with all proteins in the spot. In contrast,the beads used in the present invention can be kept fully hydrated sothe proteins are never exposed to drying. Furthermore, theantibody-antigen interaction and hence selection of positive hits occursin a fully controllable aqueous environment selected to promote nativeprotein conformation.

In-Line Quantification of Protein Per Bead

Variability of protein content on each bead can be easily accounted forin hit selection by using AmberGen's proprietary FluoroTag™ technology(FIG. S11 and described in U.S. Pat. Nos. 5,643,722; 5,922,858;6,210,941; 6,303,337; 6,306,628; 6,344,320; 6,358,689; 6,566,070;6,596,481; 6,875,592; 6,949,341; 7,169,558; 7,252,932) which allowsincorporation of fluorescent labels during cell-free synthesis.Alternatively, two-tag technology described in U.S. Pat. No. 7,423,122provides a means of probing protein content on beads using afluorescently labeled antibody directed against the N-terminal epitopeof each protein.

Fluorescence-MALDI Image Synchronization for Bead Selection

An important feature of one embodiment of this invention is the abilityto synchronize the fluorescent scan with the MALDI-MS measurements onthe bead array thereby reducing the number of beads which need to bescanned by MALDI-MS. In this embodiment, beads which exhibit aninteraction between the bait molecule residing on the bead and preymolecule is measured first using conventional fluorescent scanning anddetection methods to determine if a fluorescently labeled prey moleculehas interacted with the bait molecule on a particular bead in the array.The position of the positive hits detected in this scan are then used todirect the MALDI-MS spectrometer to the position of these positive beadsin order to determine the mass of the mass tags residing on individualbead and thus the identity of the protein. It is to be understood thatthis approach is not limited to fluorescent measurements but otherdetectable properties such absorption, luminescence, Raman, infraredcould be used to detect positive interactions of bait and prey moleculesand the positions determined used to guide measurements of the beadarray for subsequent MALDI-MS measurements and decoding.

Typically, fluorescent detection is performed with a conventionalfluorescent scanner which can assay hundreds of thousands of beads in asingle scan in a few minutes at high resolution (typically 3-5 microns).The arraying of the beads on a substrate with uniform spaced wells ofproper depth allows only one bead per well such as for the case of thepica-well plates designed for 34 micron diameter beads described abovethus preventing the overlap of beads and subsequent ambiguousmeasurements. Once the positive-hits are identified, the massspectrometer can be directed to measure the mass tags residing on thoseparticular beads identified by fluorescence, thereby avoiding themeasurement of mass tags on every bead residing in the array.

As described, elsewhere in this invention prey molecules can also bepreferentially attached to mass-tags, either directly or through bindingagents, thus providing an separate means to detect and confirm positivehits (i.e. interaction of prey and bait molecules on a bead) and also todetermine the identify of one or more prey molecules which might existin a mixture or solution and may interact with the bait moleculeresiding on an individual bead.

One embodiment of this invention utilizes beads which are bothmass-tagged and fluorescently labeled to aid in guiding the MALDI-MASSspectrometer to measure more accurately to the position of beads withpositive hits identified by fluorescence. In this embodiment, the x-yposition of the so-called synchronization beads is first determined inthe fluorescent scan. The synchronization beads comprise a fluorescentlabel and a mass tag. The fluorescent label used for synchronizationbeads has properties different from those fluorescent labels used tolabel prey molecules (e.g. a distinguishable emission spectrum). Inaddition, the mass tag(s) used for synchronization beads has a differentmass then other mass tags used to identify beads with bait molecules orprey molecules. Once the x-y coordinates of these synchronization beadsis determined from the fluorescent image of the bead array, theinformation can be used to guide the MALDI-MS instrument to find thesame beads in the MALDI-MS generated image.

Confirmation that the MALDI-MS system is measuring the correct bead inthe array is provided by measuring the corresponding mass tag residingon the synchronization beads. Importantly, once the synchronizationbeads are identified in both the fluorescent image and MALDI image, theposition of positive beads identified by the fluorescent scan can bemore accurately located in the MALDI-MS scan and mass tags measured todetermine the identity of the bait and prey molecules residing on thebead. Increased accuracy for identification of positive hits can beaccomplished by increasing the number of synchronization beads randomlyincorporated into the array, thus providing more local coordinateinformation to determine precise location of nearby positive-hits. Thismethod is especially useful for MALDI-MS systems which do notintrinsically have high resolution scanning capability (e.g. MALDI laserbeam diameter which exceeds the size of the bead diameter) or in caseswhere the positional accuracy of the wells incorporated into thepico-well plates vary in position compared to exact 2-D periodiclattice.

It is to be understood that in addition to using fluorescently labeledsynchronization beads described above other detectable properties can beused including absorbance in the visible, infrared or UV, Ramanscattering and even magnetic properties. In each case, a scan of thisproperty provides the MALDI-MS system with coordinates that aid inscanning of the beads. As one example, of how this feature might beimplemented on commercially available instruments, the BrukerUltraflextreme MALDI-MS spectrometer is equipped with software thatallows one to synchronize features obtained by scanning the sample on anexternal system based on fluorescence or absorption properties with avisual image obtained using its high resolution camera incorporated intothe machine.

In one preferred embodiment, the detection of fluorescent or otherproperties such a light absorption of the beads is measured directly onthe MALDI-MS system in order to identify positive hits (positiveinteractions between prey and bait) and then used this information todetermine which beads are measured directly by MALDIMS.

For example, the high resolution capability of the camera and imagingsystem which is incorporated into the Bruker Ultraflextreme MALDI-MSinstrument allows detection of beads of less than 20 microns that havebeen colored with a light absorbing chromogenic dye. Since a variety ofchromogenic based agents have been developed to label positiveinteractions between bait and prey molecules such as the use ofantibodies conjugated to horse radish peroxidase (HRP). A similarlabeling method can be used to directly label positive bait and preyinteractions on individual beads which can be detected directly in theMALDI-MS instrument such as the Bruker Ultraflextreme. Those skilled inthe art of MALDI-MS instrumentation will also recognize it is possibleto incorporate fluorescent detection so that fluorescent labelingmethods can be utilized to identify bait-prey interactions on individualbeads.

In addition to pico-wells formed from fiber optic bundles, aphotolithographic method of well fabrication can be utilized to increaseaccuracy of the position of each well. In addition, the ability offluorescent scanners to detect multiple wavelengths will enable markerbeads to be utilized that will allow more accurate registration of thefluorescence and MALDI images to be made.

Additional Methods of Selecting Positive Bait-Prey Interactions on Beadsfor MALDI-MS Decoding.

An alternative (or complement) to direct selection of positive hits(beads) by fluorescence imaging prior to MALDI-MS decoding is the use ofphysical methods to separate positive beads from negative beads. Onesuch approach is the use of fluorescence assisted cell-sorting (FACS). Asecond method is based on a magnetic bead sorting techniques.

Similar to conventional magnetic cell-separation techniques, theprotein-beads of Bead-GPS™ can be pre-isolated by small (1 micron)magnetic particles prior to fluorescence imaging and/or MALDI-MSdecoding. Moreover, magnetic particle manipulation is particularlyamenable to automation, for example, as achieved by Bio-Rad's (Hercules,Calif.) BioPlex multiplex immunoassay system.

Another embodiment of this invention involves the isolation of the 34micron agarose beads using fluorescence activated cell sorting (FACS).Importantly, this method is high throughput (can process millions ofbeads in a few minutes) and has the ability for greater reproducibilityand specificity than the magnetic method, since beads can be analyzed bymultiple parameters on a bead-by-bead basis. As shown in FIG. 11(Experimental Examples), blank protein beads (cell-free expressionlacking expressible DNA) and beads containing a novel autoimmuneautoantigen for PBC were separately prepared and probed with anappropriate antigen positive human serum. Bound autoantibody wasdetected with a fluorescently labeled secondary antibody (fluorescein).As seen, using the same cutoffs, 93% of the control beads were scorednegative while 96% of the autoantigen beads were scored positive.Specificity of the fluorescence signal is verified by analysis in usinga second fluorescence channel (Cy3), showing no significant signal.

Although selection either by imaging or physical separation of positivehits (beads) prior to MALDI-MS decoding is the preferred method, itshould be noted that it is not required. As demonstrated, mass-tags canbe used alone for both bead identification and autoantibody readout (seeexamples). In this case, since hits are not pre-imaged by fluorescence,the entire library is imaged by MALDI-MS in the pico-well plates.Importantly, the newer generation of faster scanning MALDI-MSinstruments can do this in a relatively short amount of time.

Mass-Tags for Bead and Prey Decoding

Basic Concept: Once beads have been sorted or selected for positive“hits” on the basis of fluorescence scanning as described above, thenext step in one preferred embodiment of this invention is decoding thebeads in order to identify the bait molecules which are bound to them. Asimilar process is also used for identifying prey molecules whichinteract with the bait molecules (see below). It is also to beunderstood that the use of fluorescence scanning in some embodiments isnot necessary in cases where each bead in the bead array is cannedindividually. In this case, positive hits can be identified usingdecoding methods described in this invention.

One preferred embodiment of the invention which entails a method ofdetecting the interaction of prey molecules with bait molecules,comprises: a) providing a mixture comprising first and second beads,said first bead comprising a first mass tag and a first bait molecule,said second bead comprising a second mass tag and a second baitmolecule, wherein said first and second bait molecules and said firstand second mass tags are different; b) making an array with said beads;c) contacting said first and second bait molecules with a solutioncomprising a prey molecule, wherein said prey molecule comprises a masstag; and d) subjecting said array to MALDI mass spec analysis underconditions wherein binding of said prey molecules to a bait molecule isdetected.

A second embodiment of the invention involves reversing steps b) and c)listed above so that the contacting of said and first and second baitmolecules with a solution comprising a prey molecule occurs beforemaking an array of said beads.

It is to be understood that the bait and prey molecules can consist of alarge variety of different biomolecules or biologically active moleculessuch as proteins, antibodies or potential drug compounds. For example,bait or prey molecules includes but are not limited to proteins,polypeptides, nucleic acid molecules, lipids, carbohydrates,biologically active drug compounds, hormones, antigens, antibodies andcombinations of these molecules. The aforementioned bait and/or preymolecules can also be labeled using standard fluorescent labelingreagents comprising one or more fluorophores (see examples) in order toidentify positive bait/prey interactions and facilitate fluorescentimage synchronization with MALDI-MS imaging as described elsewhere inthis invention.

In one example, bait molecules consist of various proteins selected froma protein library such as can be expressed as described in thisinvention using the commercially available 12,000-member Open ReadingFrame (ORF) template library (ORFeome), whereas prey molecules consistof autoantibodies present in a patient's blood that are associate withautoantigens underlying an autoimmune disease such as primary biliarycirrhosis or lupus. Similarly, antigens might be tumor specific (tumorassociated antigens (TAAs) related to a particular cancer tumor andantibodies freely circulating in blood may be formed in response tothese TAAs and used as biomarkers for detection of the cancer or topredict the course of the cancer (prognostic). Detecting and identifyingthe interaction of specific antigens with specific antibodies using thepresent invention provides critical information in designing diagnostictests, prognostic tests and therapeutic methods related to thesespecific autoimmune diseases and for specific cancers.

In another example, bait molecules consist of various proteins selectedfrom a protein library such as expressed from the commercially available12,000-member Open Reading Frame (ORF) template library (ORFeome),whereas prey molecules consist of small molecules that have beenselected using standard screening methods well known in thepharmaceutical industry as potential drug compounds. Alternatively theprey molecules might be part a small compound library used to screen forpossible drug candidates or drug targets. Detecting and identifying theinteraction of a library of small compounds and library of proteinssimultaneously using the present invention provides critical informationfor the pharmaceutical industry in identifying potentially useful drugs,drug targets and also to identify side-effects of drugs.

In another example, both bait and prey molecules consist of variousmolecules selected from a protein library such as expressed from thecommercially available 12,000-member Open Reading Frame (ORF) templatelibrary (ORFeome) proteins. In this case, the detection andidentification of specific protein-protein interactions providesimportant information for elucidating various cellular pathways and therole that specific proteins play in active cellular process and indisease. In addition, this information can lead to the discovery of newbiomarkers for diagnosis and new drug targets to treat specific diseasesinvolving these cellular pathways.

In another example, bait molecules consist of various proteins selectedfrom a protein library such as expressed from the commercially available12,000-member Open Reading Frame (ORF) template library (ORFeome) whichhave been treated with a biologically active molecule which produces achemical or structural change in particular proteins or polypeptides inthe library, whereas prey molecules consist of molecules which aredetect or probe chemical and structural changes in the bait molecules.As one example, the protein bait molecules is treated with a specifickinase which causes phosphorylation of specific Tyr, Ser or Thr residuespresent in the sequence of specific proteins or polypeptides. Once theproteins are treated with this kinase, antibodies that are specific forphosphorylated Tyr, Ser or Thr which constitute the prey molecules areallowed to interact with said protein bait molecules. Detecting andidentifying the interaction of specific antibodies with thephosphorylated proteins provides important information about kinasesubstrate specificity and can identify new drug targets and drugs totreat specific diseases.

In another example, bait molecules consist of various antibodiesselected for their specific interaction with a target analytes such as aparticular proteins or other molecules whose concentration in a sampleis to be determined, whereas prey molecules consist of variousantibodies selected for their specific interactions with the same set oftarget analytes. In this case, detecting and identifying the interactionof bait and prey via mutual interaction with the analyte providesimportant information about the analytes presence in the sample and itsconcentration. Such a “sandwich” configuration of antibodies directedagainst a target analyte in a mixture to be measured is commonly used insandwich ELISAs and is well known to those in the field of biochemistryand molecular biology. In this case, using the methods provided in thisinvention, the concentration of thousands of analytes in a sample can besimultaneously measured. In addition, using methods described for codingbait and prey with mass tags, the expression level of thousands ofanalytes in multiple samples can be determined.

It is to be understood in addition that the mass tags used to code baitor prey molecules can consist of a wide variety of molecules and theirisotope labeled variants including but not limited to polypeptides,oligonucleotides, linear block co-polymers, branched polymers and smallmolecules such as those part of a small compound library used to probedrug targets.

In one embodiment, the method of decoding is based on the use ofmass-tags and more preferably photocleavable mass-tags which remaincovalently attached to the bead or attached via a binding agent untilexposed to light (see below). In one preferred embodiment of thisinvention, the mass-tags are modified polypeptides whose sequence hasbeen chosen so that its mass is unique (i.e. differs from every othermass tag used in the library). In a second embodiment, the mass-tags areisotopically labeled molecules with the same structure but differentmasses. In a third embodiment, the mass tags consist of a differentpolymers than a polypeptide such as an oligonucleotide. In a forthexample, a 2,2′-(ethylenedioxy)-bis-(ethylamine) is used as the basicbuilding block for constructing the mass tag. It is to be understoodthat in this invention there are a variety of molecules which wouldserve as mass tags and it is not limited to one class of molecule orpolymer.

In the case of polypeptides or modified polypeptides which serve as masstags it is to be understood that a relatively small peptide (e.g. anoctamer, N=8) can provide sufficient number of sequences to providesufficient unique masses to satisfy even a large-library of 100,0000different proteins (20N=˜25×109). In practice, the number of viablesequences depends on the mass resolution of the MALDI-MS instrumentwhich is often better than 0.1 daltons in the mass range measured. Inaddition, any degeneracy in the molecular weight of the mass tags can bedecoded using the ability of MALDI-MS to sequence small peptides (<5,000MW), commonly know as MS-MS-TOF. Additional “fine-tuning” of masses canbe accomplished by modification of the mass-tag such as the addition offluorescent labels.

As shown in FIG. S04, multiple-mass tags can be deployed on each bead todetermine the identity of the attached protein (red), the sample beingscreened in cases where multiple samples are scanned (sometimes referredto as bar-coding) (purple) and the presence of an interacting antibodyindicating a positive hit or biomarker (green). Since, as describedabove beads have been already selected by fluorescence scanning on thepico-well slide, this last mass-tag serves to reduce false-positivesensuring higher accuracy for biomarker selection.

A single mass tag of sufficient length or multiple mass tags can be usedto code a bead set which is contacted with a solution of prey moleculesfrom a set of multiple solutions in order to determine the presence ofprey molecules in each solution. In this case, solution 1 containing aset of prey molecules is mixed with beads coded with mas tags that areunique for that set of bait molecules and solution 2 containing adifferent set of prey molecules is mixed with the beads coded with adifferent mass tags that uniquely coded that set of bait molecules. Thebeads from these two steps after contact with the respective solution 1and 2 containing prey molecules are then mixed together and used to forma random array. The fact that different sets of mass tags are used tocode the two sets of beads allows the prey from solution 1 whichinteract with bead set 1 and the prey from solution 2 which interactwith bead set 2 to be uniquely determined. It will be understood bythose familiar with the barcoding approach applied in genomic DNAsequencing that such an approach will allow prey interaction with baitto be determined uniquely for a large set of samples.

Photocleavable Linkers for Mass Tags

In some embodiments where mass-tags are attached to beads foridentification of bait and/or directly to prey molecules, the mass tagsdo not need to be directly covalently linked to the bead surface or preymolecule but instead bound through a binding agent such as anantibody-polypeptide interaction (e.g. Experimental Example 3). However,this is non-ideal since stringent wash steps can result in partialremoval of the tags (as observed during the course of some ourexperiments). One solution to this problem is covalently attachedmass-tags which are photo-released upon exposure to UV-light.Alternatively, a near-covalent strength linkage between (strept)avidinand biotin (Kd=10⁻¹⁵) can be used in conjunction with a photocleavablelinker (e.g. Experimental Example 5).

AmberGen has developed a novel class of photocleavable linkers(PC-Linkers) useful in a variety of applications such as photocleavageassisted molecular purification, tRNA-mediated protein engineering,photo-activation of compounds, biomolecules and viruses as well asphotocleavable mass-tagging for multiplexed assays [Olejnik,Krzymanska-Olejnik et al. (1996) Nucleic Acids Res 24: 361-6; Olejnik,Krzymanska-Olejnik et al. (1998) Nucleic Acids Res 26: 3572-6; Olejnik,Ludemann et al. (1999) Nucleic Acids Res 27: 4626-31). In the case ofmass-tagging of the proteomic bead-library, a short peptide with 7 or 8amino acids is linked to the beads via a photocleavable linker. Notethat previous experiments have demonstrated that AmberGen's PC-Linker israpidly photocleaved with 95% efficiency is less than 10 minutes using alow-intensity commercial black-light [Olejnik, Ludemann et al. (1999)Nucleic Acids Res 27: 4626-31].

In one embodiment of the invention PC-Mass-Tags for proteinidentification are attached to beads in either one of 2 ways asillustrated in FIG. S05 and detailed below:

Ultra-High Affinity Biotin-(Strept)Avidin: Peptide mass-tags modified atthe N-terminus with AmberGen's photocleavable biotin are attached to(strept)avidin coated beads (see FIG. S11 for attachment options of thecell-free expressed proteins). This mass-tagging method has already beendemonstrated in the Experimental Examples (FIG. 5).

Direct Covalent: Using the primary amine-reactive NHS chemistry on theuncoated agarose beads, peptide mass-tags bearing an N-terminalphotocleavable primary amine moiety will be chemically attachedsimultaneously with the attachment of the protein capture element (e.g.capture antibody). This is highly analogous to AmberGen'sphosphoramidite technology distributed through Glen Research Inc. forintroducing a photocleavable primary amine at the 5′ end of DNA[Olejnik, Krzymanska-Olejnik et al. (1998) Nucleic Acids Res 26:3572-6]. For this method, peptide mass-tags lacking lysines (reactiveprimary amine on side chain), or where lysines are blocked on the6-amine, will be used to avoid non-cleavable attachment to theNHS-activated agarose beads.

A library of peptides pre-screened by mass spectrometry could beobtained by commercial synthesis from a variety of available vendorssuch as Mimotopes (Austria), Peptide 2.0 Inc. (Chantilly, Va.) orGenScript Inc. (Piscataway, N.J.) and used to create the mass-tags whichwill be photocleavably linked to the beads. High throughput peptidesynthesis services are available from these vendors (e.g. solublepeptide arrays in 96-well plates) and peptides can be purchased withfull. HPLC and mass spectrometry quality controls. Conventionalsolid-phase chemical peptide synthesis begins at the C-terminus and endsat the N-terminus. The growing peptide is tethered to the solid-phasesynthesis resin via its C-terminal carboxyl group, exposing itsN-terminal amine (after deprotection) and allowing sequential attachmentof another N-terminal blocked amino acid precursor (again followed bydeprotection). Thus, the attachment of N-terminal modified PC-Biotin orPC-amine (amine protected) amino acid precursors at the final cycle ofsynthesis is a relatively strait forward process.

We have calculated that due to the high analytical sensitivity of massspectrometry (attomoles), even adding 10 fmoles per bead of mass-tags(10-mer), the aforementioned peptides with N-terminal PC-Linkermodification and all quality control data will add only pennies (¢10) tothe cost of an entire proteome-bead library.

In addition to PC-Mass-Tags attached to the beads for identificationpurposes, Bead-GPS™ utilizes PC-Mass-Tags attached to the probes used toquery the proteome library. In the case of autoantigen discovery, thePC-Mass-Tag is attached to the anti-human IgG secondary antibody used todetect the bound serum autoantibody. In this case, only one species ofunique mass-tag is required. This has already been demonstrated inExperimental Example 7 (FIG. 7) using PC-Biotin. In one embodiment,custom reagents can be synthesized to allow direct covalent labeling ofprobes (e.g. antibodies) with PC-Mass-Tags (FIG. S06).

Mass-Tag Decoding

In general, a requirement of mass-tag decoding is that each mass-tagpeak must correspond to the correct molecular weight predicted by themass tag molecular structure such as for example a given polypeptidesequence plus any modifications or isotope labeling within theresolution of the spectrometer in the specific mass range (˜0.1 Da in600-4,000 Da range).

It is highly desirable that each mass-tag peak must have asignal-to-noise ratio of at least 50:1, although lower signal-to-noiseis sufficient for some applications. For comparison, the signal-to-noiseratio of single prototype mass-tags attached to beads incorporated intoordered arrays as described in the examples are routinely greater than250:1 using a set of standard mass spectral parameters. Note that thesignal-to-noise ratio in all experiments is determined using the ABI4800software, which measures the integrated target peak intensity and ratiosthis to the integrated intensity of a nearby background region whichexhibits no detectable peaks.

Importantly, spectral resolution and mass accuracy of the ABI 4800 PlusMALDI-TOF-TOF analyzer is sufficient to unambiguously identify peptidesseparated by as little as 0.1 Da. However, one potential problem is theappearance of several peaks for each peptide in the mass spectra, whichare separated by 1 Da (the “isotope envelope”), due to the presence ofsmall amounts of mass-shifted C13 and N15 atoms in the protein sequence.In the case of two mass tags separated by only a few Daltons, thespectral overlap may affect the tag identification. This will beaddressed by using, in real-time, a spectral processing routine calledpeak de-isotoping. The routine, which is built-in into the ABI 4800 dataacquisition software, replaces multiple peaks in the isotope envelopewith a single mono-isotopic peak (corresponding to the sequencecontaining only C12 and N14 atoms).

MALDI-MS Imaging Software

The MALDI-MS imaging of individual beads described in this inventionrequire software to analyze data and to identify mass-tags on individualbeads. There are a variety of software packages available commerciallyfor this purpose. As an example, we have utilized BioMap in theExperimental Examples, which is a powerful biomedical image analysissoftware package supporting various data types generated by optical,PET, CT and mass-spectrometry based imaging. The BioMap platform allowsvisualization and storage of large volumes of data includingexperiment-specific information such as scan ID, experimental protocoland sample history. It is also a flexible tool that can be easilymodified to accommodate a specific requirement. It is contemplated thatas part of this invention improved imaging of individual beads andmass-tags can be made that is designed for MALDI-MS bead-imagingworkflow, such as automated co-registration of fluorescent and MALDI-MSscan images and identification of positive “hits” based on the detectionof PC-Mass Tags.

Automation

In general, mass spectrometry and MALDI-MS in particular have proven tobe highly amenable to high throughput applications in both clinical andbasic research settings. For example, Sequenom Inc. has establishedMALDI-MS as an effective technique in the field of genotype profiling,and is providing diagnostic products in this area. As a second exampleof automation of mass spectrometry in clinical diagnostics, thePediatrix Medical Group, the largest provider in the US for neonatalblood tests, uses tandem array mass spectrometry to detect metabolicdisorders and has screened over 2 million babies using this method.

In the case of this invention, many improvements are envisioned whichcan facilitate automation and high throughput biomarker discovery. Forexample, multiplexing can be achieved at several stages including duringthe preparation of the bead library and in bar-coding multiple sample.Importantly, the use of a highly automated mass spectrometer such as theABI 4800 Plus MALDI-TOF MS or the more advanced ABI 5800 will alsofacilitate high throughput analysis at the MALDI-MS bead scanning stage.For example, this system uses advanced software designed for automatedscanning of a two-dimensional area, data collection and spectralprocessing. The ability to automatically scan approximately 10,000 beadsin the pico-well MALDI plate in one hour is possible. Use of the ABI5800 should reduce this time to 1/10 (6 minutes). Furthermore, using oneof the commercially available plate-loading robots will allow use of theinstrument in the operator-free mode, 24 hours a day. As an example ofautomation levels achievable with MALDI, Sequenom, Inc. has introduced aMALDI-based system for SNP analysis which is capable of analyzing100,000 genotypes per day.

In Situ Mass-Fingerprinting of Proteome Bead-Arrays

Rather that the addition of exogenous mass-tags, or any other tags, itis possible to utilize the bead-bound cell-free expressed human proteinsthemselves as identification codes. Analogous to mass spectrometry basedon mass-fingerprinting used in classical proteomics, proteins aredigested with protease (e.g. trypsin) and the resultant peptide“fingerprint” used for protein identification. If necessary, thepeptides are further fragmented in the TOF/TOF tandem mass spectrometerand sequenced using the standard capabilities of today's instruments. Wehave explored this possibility using in situ trypsinization ofprotein-bead arrays in the pico-well plates. In the experiment shown inFIG. S13, human p53 and GST A2 beads were produced using theaforementioned methods (antibody-mediated C-Tag capture; 75-100 micronbeads in this case). Following deposition into a MALDI plate, the beadswere sprayed with an ultra-fine mist of trypsin solution and allowed todigest in a humidified chamber. The reaction was stopped by spraying onthe MALDI matrix solution (denaturing). Individual beads were thenimaged by MALDI-MS. In this case, MALDI images were generated based onsingle identifying tryptic fragments (confirmed by tandem MS/MSsequencing) corresponding to the p53 and GST proteins. See alsoExperimental Example 9 (FIG. 9).

Application to Detection of Genetic Mutations using MALDI-MS-Imaging,Mass Tags and Arrays of Beads

One embodiment of this invention is directed towards the multiplexdetection of mutations which might exist in multiple regions of singlegenes or multiple genes. In order to detect such mutations, a nascentprotein or polypeptide (typically a portion of a gene product, whereinthe portion is between 5 and 200 amino acids in length, and morecommonly between 5 and 100 amino acids in length, and more preferablybetween 5 and around 60 amino acids in length—so that one can work inthe size range that corresponds to optimal sensitivity on most massspectrometry equipment) is (in one embodiment) first synthesized in acell-free or cellular translation system from message RNA or DNA codingfor the protein which may contain a possible mutation. The nascentprotein or polypeptide is then separated from the cell-free or cellulartranslation system using an N-terminal (located at or close to theN-terminal end of the protein) which is designed to bind to a bindingagent on the surface of a bead. For example, the C-terminal epitope canconsist of an HSV sequence as discussed here and binding agent on thebead consists of an anti-HSV directed antibody. A C-terminal epitope isnot used to avoid the case of chain truncating mutations which wouldeliminate this epitope.

This process as described above can then be repeated to examineadditional sequences in a given gene or multiple genes. In cases wheregenomic material is used this may be necessary in order to span wholeexons or pieces of exons such as in the BRCA1 or BRCA2 gene whichcontains over 50 exons. Alternatively, different sequences in differentgenes may wish to be examined in the case for example of a tumor wheremultiple oncogenes may be suspect. Thus, using the methods described inthis invention, a library of beads will be formed each one containing asequences derived from different gene sequences and also containingunique mass tags coding that particular type of bead. In this case, thesequence interrogated on the individual bead may be a mixture ofwild-type sequence and mutant sequences derived from the same region ofthe gene interrogated.

The resulting isolated material (which may contain both wild-type andmutant peptide sequences) is then analyzed by mass spectrometryconsisting of the measurement of individual beads which are part of abead array. Detection of a peak in the mass spectrum with a masscorrelating with the expected wild-type peptide indicate the wild-typepeptide. Detection of a peak in the mass spectrum with a mass notcorrelating with the wild-type peptide indicates a mutation.

It is important to note that in this example, the mass of the wild-typesequence and the resulting peak it produces from an individual beadserves the role of a mass-tag, e.g. it allows one to identify the baitspecies captured on the bead. The presence of a mutation is thenidentified by the additional peaks from the bead which do not correspondto the wild-type species. For example, a missense change of a wild-typesequence which corresponded to a codon shift from TAT to TCT wouldresult in the substitution of a Tyr with a Ser and a subsequent massshift of +176 daltons. It will be understood by those practiced in theart of mass spectrometry that advanced systems are able to distinguishmuch small shifts even below 1 dalton so almost all substitutions can bedetected. Furthermore, MS-MS techniques allow sequencing of the peptidesto resolve any ambiguity if the wild-type peptide is not unambiguouslyidentified by its mass. In some embodiments it may be advantageous toalso code the bead containing a particular polypeptide species usingmass tags as described extensively in this invention. For example, inthis embodiment the molecules capture with a binding agent on the beadsurface consist of nascent proteins or polypeptides synthesized in acell-free or cellular translation system from message RNA or DNA codingfor the region which may contain a possible mutation. Furthermore, thenascent proteins produced in the cell free reaction could be added inseparate reactions to a particular mass-tagged bead as describedpreviously under parallel method or formed using the batch techniquesdescribed before.

It is to be understood that the protein bead library used in thisembodiment could be formed using the Parallel Synthesis methodsdescribed previously or the Batch Synthesis methods describedpreviously. In the Parallel Synthesis methods each protein orpolypeptide sequence is synthesized using PCR and cell-free synthesis inseparate reactions and the resulting proteins added to individualsuspensions of beads whereas in the Batch Preparation method, the entirelibrary can be formed in two multiplex reactions. In either case, theoverall library is randomly arrayed and the individual beads measuredusing mass spectrometry.

Photocleavable DNA-Tags

In addition to peptide based decoding of the proteome-bead library, wehave developed an alternative method of coding individual beads based onthe use of PC-DNA-Tags. Such tags are also based on proprietaryphotocleavage technology developed by AmberGen (see U.S. Pat. Nos.5,948,624; 5,986,076; 6,589,736; 7,312,038; 7,339,045; 7,211,394;6,057,096; 6,218,530; 7,057,031; 7,195,874; 7,485,427; 7,547,530) whichoffers convenient synthesis of DNA molecules with a 5′-modificationconsisting of a photocleavable linker such asPC-aminotag-phosphoramidites commercially distributed by Glen ResearchInc (Sterling, Va.). These tags can be directly linked to the activatedagarose beads similar to the method used for PC-Mass-Tags and releasedupon exposure to near-UV light. Once removed from individual positivebeads, these PC-DNA-Tags can be rapidly decoded and quantified in bulkusing a massively parallel PCR platform.

One embodiment of this invention involves use of photocleavable DNA-tagsto code and decode beads. As an example, solid-phase (bead) PCR withuniversal photocleavably attached primers, can be used to separatelyamplify various human ORF plasmid inserts on a 34 micron agarose beads;thus creating photocleavably tethered DNA amplicons (pure species oneach bead). Several different DNA-bead species are then pooled atvarious ratios and then photocleaved.

The photo-released DNA is then analyzed on a suitable instrument whichcan detect the DNA-tags such as a standard DNA hybridization chip (e.g.DNA microarray) or an RT-PCR device. In the case of DNA hybridizationchips, many chips are available such as from Affymetrix which haveprobes for thousands of genes that can be used to detect the release ofspecific DNA sequences photoreleased from the beads. Commercialprototypes have also been introduced such as by WaferGen Inc. that cansimultaneously analyze large numbers of such PC-DNA tags. In oneexample, a 5,000-member prototype RT-PCR chip was used containing probesto all members of the test bead library evaluated. As shown in FIG. 10(Experimental Examples), gene ORFs were positively identified from aslittle as a single bead, with Cycle Threshold (Ct) values approximatelyfollowing the bead numbers.

Importantly, attachment of PC-DNA-tags is fully compatible with theParallel protein-bead library production methods described above.Photocleavage of the individual beads and collection of DNA-tags frompositive beads can be easily accomplished almost simultaneous withfluorescent scanning (i.e. bead selection step) by using a modifiedfluorescence microarray scanner. In particular, a laser normally usedfor scanning the image can be replaced with a laser capable ofphotocleavage of DNA from individual beads such as a pulsed Nd—Yag laserwith 355 nm output which are widely commercially available at low cost.It has been demonstrated by us that such lasers can photocleave >90% ofthe tags on a bead sample in less than a few seconds. Since commercialfluorescent scanners operating with multiple wavelengths and differentlasers are designed to perform sequential scans maintaining imageregistration, software image identification of positive beads wouldallow the Nd—Yag laser to be switched on to expose only positive beadsduring a sequential registered scan. Scan resolution is normally 3-5μallowing high precision for Nd—Yag laser beam to photocleave DNA-tagsfrom 35μ beads located in the pico-well plate. Alternatively a scannerusing a CCD imager along with a photocleaving laser can be readily usedto selectively remove DNA-tags from specific beads identified aspositive in the fluorescent scan. Photocleaved DNA-tags can be collectedin a thin fluidic chamber overlaying the array for subsequent decoding.Importantly, selection of the positive hits is simplified for thisapproach since the imaging and photo-release are done simultaneously inthe same instrument.

Importantly, both the in situ trypsinization and PC-DNA based decodingapproaches are also fully compatible with the Batch method ofprotein-library construction described earlier which involvessingle-molecule solid-phase emulsion PCR to create a bead-sorted libraryof expressible DNA in a single reaction, which is then converted to abead-sorted library of in vitro expressed proteins in a singleself-assembling cell-free protein synthesis reaction.

EXPERIMENTAL Example 1 Affinity Purification of Cell-Free ExpressedPeptides onto an Agarose Bead Affinity Resin Followed by MassSpectrometry Detection from Single Beads

In this Example a test peptide corresponding to a segment of the APCgene, with an expected molecular weight of 6,203 Da, containing anN-terminal FLAG® epitope tag (Sigma-Aldrich, St. Louis, Mo.), wassynthesized in a recombinant cell-free transcription/translationreaction according to the manufacturer's instructions (PureSystem; PostGenome Institute Co., LTD., Japan).

The nascent peptide from the reaction was then purified on mouseanti-FLAG antibody-coated agarose affinity beads (˜75-150 microndiameter). For this, the crude cell-free expression mixtures werediluted with 50 μL of AB-T [100 mM ammonium bicarbonate with 0.1% TritonX-100 (v/v)]. Mouse anti-FLAG antibody coated agarose affinity beadswere used in batch mode to purify the cell-free expressed peptides(EZview™ Red Anti-FLAG® M2 Affinity Gel, Sigma-Aldrich, St. Louis, Mo.).The diluted crude cell-free expression mixtures were combined directlywith ˜1 μL beads in 0.5 mL polypropylene PCR tubes. The mixtures werethen incubated for 20 minutes at room temperature with gentle mixing tokeep the beads suspended. The beads were then spun down in amicro-centrifuge (˜16,000×g) and the fluid supernatant removed anddiscarded. The beads were then washed 2×10 min each in mass spectrometrygrade water (MSG-Water), removing the fluid supernatant as before. Afterremoving the final wash, beads were re-suspended in 50 mL MSG-Water andindividual beads were selected from suspension by careful pipetting anddeposited onto a stainless steel plate for matrix assisted laserdesorption ionization time-of-flight mass spectrometry (MALDI-TOF orMALDI-MS). A small volume (0.2-0.5 μL) of MALDI-TOF matrix solution (20mg/mL sinapinic acid matrix in 50% acetonitrile and 0.1% trifluoroaceticacid) was immediately applied directly on top of the beads. The dropletwas then allowed to dry/crystallize under ambient conditions withoutdisturbance. The size of the final spot was approximately 2 mm indiameter with the beads near the center of spot. Once completely dried,the spots were analyzed using a Voyager-DE MALDI-TOF mass spectrometer(Applied Biosystems; Foster City, Calif.). The MALDI-TOF spectra wereacquired on the outer edge of the spot, inside the spot in the immediatevicinity of the beads and also directly from the beads.

Results:

FIG. 1 shows that the peptide was observed at the correct mass of 6,203Da (mass includes N-terminal formylation produced in the cell-freeexpression system). These data confirm that the amount of peptide thatcan be bound to single agarose beads of roughly 100 microns in diameter,is sufficient to be detected by MALDI-TOF mass spectrometry. This isconsistent with the reported capacity of the agarose beads, whichat >100 ng/μL beads and approximately 1,000 individual beads per μL beadvolume, would amount to approximately 20 femtomoles of a 6,000 Dapeptide. This falls within range of the sensitivity of MALDI-TOF massspectrometry. The signal intensity was typically higher near the beads,although the matrix solution can elute peptides from the beads resultingin peptide spreading prior to drying of the matrix solution spot.

Example 2 Mass Spectrometry Readout and Mass-Imaging from IndividuallyResolved Beads

In this Example, two test peptides with molecular weights of 3,483 Daand 3,287 Da, each containing an N-terminal FLAG® epitope tag(Sigma-Aldrich, St. Louis, Mo.), were separately synthesized in arecombinant cell-free transcription/translation reaction according tothe manufacturer's instructions (PureSystem; Post Genome Institute Co.,LTD., Japan). The nascent peptides from each reaction were thenseparately purified on mouse anti-FLAG antibody-coated agarose affinitybeads (EZview™ Red Anti-FLAG® M2 Affinity Gel, Sigma-Aldrich, St. Louis,Mo.) (˜75-150 micron diameter). The beads were then mixed in a 9:1 ratioand manually deposited in a random pattern on a suitable substrate forMALDI-TOF mass spectrometry. The MALDI-TOF matrix (CHCA) was sprayed onin a thin and uniform film and allowed to dry. MALDI-TOF imaging of thesurface was performed using an ABI 4800 Plus MALDI-TOF/TOF massspectrometer (Applied Biosystems; Foster City, Calif.). The surface ofthe substrate was scanned with the instrument's laser, in theinstrument's reflector mode, in the 1,500-4,000 m/z (mass/charge)spectral range and two images were constructed using spectral intensityat the m/z corresponding to the molecular weight of the peptides.

Results:

A two-color image overlay was created from the two mass-images of thebeads that were constructed using the spectral intensity at the m/zcorresponding to the molecular weight of the two test peptides (FIG. 2).Two distinct and resolved populations of beads are visible on thesubstrate, with the ratio of the two bead species in the mass-imagebeing in excellent agreement with the mixing ratio. The spectralanalysis of each spot reveals a single strong MALDI-TOF peak, indicatingthat each bead carries a homogenous population of one peptide. This workverifies the ability of MALDI-TOF mass spectrometry to scan and imageindividual beads that carry compounds (peptides in this embodiment)detectible by their unique mass.

Example 3 Mass Spectrometry Readout and Mass-Imaging from IndividuallyResolved 34 Micron Beads Deposited in Pico-Well Plates: Mass-Tagging ofAnalyte-Bearing Beads for Identification

In this Example, 34 micron agarose beads were conjugated to an antibodydirected against the HSV epitope tag. The beads were then loaded withdifferent recombinant proteins bearing this HSV epitope tag. The beadswere additionally bound to one of three different peptide “mass tags” ofunique mass, corresponding to the HSV peptide epitope itself, conjugatedon the N-terminus to different fluorophores. The beads were thendeposited into a special pico-well plate and mass-imaged by scanningMALDITOF mass spectrometry. Separately, the presence of the recombinantproteins was detected on the same batch of beads by probing with afluorescently labeled antibody directed against the common VSV-G epitopetag, also present in the recombinant proteins.

Development of Pico-Well Plates for MALDI-TOF Bead Scanning

In order to create mass images of beads or particles, it may beadvantageous to randomly array the beads in a regular two-dimensionalgrid, similar to spots in a conventional microarray. This maximizes beaddensity, yet assures bead separation, and allows the MALDI-TOFinstrument to efficiently and rapidly move from one bead to another, foroptimal scan speed. Furthermore, in order to maximize bead resolution,it may also be advantageous to contain each bead in its own microscopicwell. Finally, cross-platform imaging of the beads, such as by massspectrometry and fluorescence, may be advantageous in certainembodiments of the technology.

For this purpose, we developed a novel dual-use pico-well substratesuitable for both mass spectrometric and light based analyses. Thissubstrate, whose overall dimensions are 75.0 mm long by 25.0 mm wide and1.0 mm thick, is sliced from a fiber optic bundle (block of fused opticfibers) and fabricated by the etching of 44 micron diameter by 55 microndeep pico-wells (i.e. picoliter scale volume) at the ends of the opticalfibers that are positioned 50 microns from center-to-center in ahexagonal ordered array (Incom Inc., Charlton, Mass.). The resultis >0.5 million wells in the dimensions of a standard microarray ormicroscope slide (75×25×1 mm). The design allows deposition of only onebead per well, but maximum access of the MALDI-TOF laser beam tovaporize the matrix coating the bead and allow mass analysis. Since thearray is fabricated from a fiber optic bundle, it also forms aface-plate for convenient measurement of light based signals, forexample fluorescence or luminescence from each bead (each well) usingdirect-contact CCD cameras or by using conventional fluorescencemicroarray scanners.

Preparation of Anti-HSV Antibody Coated 34 Micron Agarose Affinity Beads

34 Micron diameter agarose beads were conjugated to an anti-HSV tagantibody for later use in capturing peptides bearing this epitope tag.To do so, an anti-HSV monoclonal antibody (EMD Biosciences, Inc., SanDiego, Calif.) was diluted to 0.5 mg/mL in a final buffer of 200 mMsodium bicarbonate and 200 mM NaCl (Binding Buffer). 6% cross-linkedNHS-activated 34 micron agarose beads (NHS HP SpinTrap, GE HealthcareLife Sciences, Piscataway, N.J.) were washed 4× in several bead volumesof ice cold 1 mM HCl. Beads were then reacted with the anti-HSV antibodysolution at a ratio of 6 μg of antibody per each μL of actual beadvolume for 1 hour with gentle mixing.

Beads were then washed 1× briefly and 2×30 min with several bead volumeseach of 200 mM glycine and 1 mM EDTA in Binding Buffer. Beads were thenwashed 2×5 min in Binding Buffer, 2×5 min in 10 mM Tris, 1 mM EDTA, pH 8with 200 mM NaCl, and 1× briefly in 10 mM Tris, 1 mM EDTA, pH 8 with 50mM NaCl. Beads were then prepared to a 20% (v/v) suspension in 10 mMTris, 1 mM EDTA, pH 8 with 50 mM NaCl and stored at +4° C.

Preparing Modified HSV Peptide Mass Tags of Different Mass

Peptides of unique mass were prepared by chemical modification withdifferent fluorophores of the HSV-Tag peptide (KQPELAPEDPED), which waspurchased from Sigma-Aldrich (St. Louis, Mo.). To do so, the peptide wasprepared to 5 mg/mL in 100 mM sodium bicarbonate and reacted overnight(with mixing) with equimolar amounts of the Cy3-NHS or Cy5-NHS activated(primary amine reactive) fluorescent dye labeling reagents (GEHealthcare Life Sciences, Piscataway, N.J.). Peptides were used withoutfurther purification (MALDI-TOF analysis showed that the peptides werealmost exclusively labeled at a ratio of 1 dye per peptide molecule).Because the NHS activated labeling reagents react only with primaryamines, selective labeling of the N-terminal lysine is anticipated. Theunlabeled as well as Cy3 and Cy5 labeled HSV peptides provided threespecies of unique mass tags, 1,368 Da, 2,048 Da and 2,074 Darespectively, which were used in subsequent steps.

Binding of Recombinant Proteins and HSV Peptide Mass Tags to theAnti-HSV Agarose Affinity Beads

Human p53 and human KLHL12 were expressed recombinantly in a cell-freereaction. Expression reactions were performed using atranscription/translation coupled rabbit reticulocyte lysate system(TNT® T7 Quick for PCR DNA; Promega, Madison, Wis.) according to themanufacturer's instructions. The expression plasmid used was aderivative of the pETBlue-2 vector (EMD Biosciences, Inc., San Diego,Calif.) containing a C-terminal polyhistidine (HHHHHH) and HSV epitopetag (QPELAPEDPED) as well as an N-terminal VSV-G epitope tag(YTDIEMNRLGK) flanking the Open Reading Frame (ORF) inserts of human p53or human KLHL12. A parallel negative control expression reaction wasperformed lacking only the plasmid DNA.

After the expression reaction, the nascent proteins were isolated ontothe aforementioned anti-HSV antibody coated agarose affinity beads.Protein isolation onto the beads was performed in batch mode using 0.45micron pore size, PVDF membrane, micro-centrifuge Filtration Devices tofacilitate manipulation of the small volumes of affinity beads andexchange the buffers (Ultrafree-MC Durapore Micro-Centrifuge FiltrationDevices, 400 μL capacity; Millipore, Billerica, Mass.). FiltrationDevices were used unless otherwise stated. For each sample (1 FiltrationDevice per sample), 1 μL packed bead volume (˜30,000 beads) was washedbriefly 4×400 μL with TBS [TBS=50 mM Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol) pH 7.5 and 200 mM NaCl] andthen 2×400 μL briefly with high purity Molecular Biology Grade Water(MBG-Water). Washed bead pellets were then re-suspended at a ratio of 50μL of crude expression reaction per μL packed bead volume (˜30,000beads) and mixed for 30 min to capture the target nascent recombinantprotein. Beads were then washed briefly 3×400 μL with TBS-T [TBS with0.05% v/v Tween-20] and then 1×400 μL with Block Buffer [1% w/v BSA inTBS-T].

Next, the beads (which at this stage already contained the nascentrecombinant proteins; 1μL packed bead volume each sample) wereadditionally loaded with the aforementioned HSV peptide mass tags. 730pmoles of the aforementioned HSV peptide mass tags was added to each ofthe three bead samples (blank, human p53 and human KLHL12 bead samples),one unique mass tag species per bead sample, in 200 μL of Block Buffer.This corresponded to ˜25 fmoles of HSV peptide mass tag added per bead.HSV peptide mass tags were allowed to bind for 30 min with mixing. Beadswere then washed 3×400 μL briefly with TBS-T, 3×400 μL TBS and then4×400 μL with Mass Spectrometry Grade Water (MSG-Water).

MALDI-TOF Mass Spectrometry Imaging of Beads

As a result of the previous steps in this Example, the three mass-taggedbead species were as follows: Blank beads (no recombinant protein) codedwith the unlabeled HSV peptide mass tag (1,368 Da), human p53 beadscoded with the Cy3 labeled HSV peptide mass tag (2,048 Da) and humanKLHL 12 beads coded with the Cy5 labeled HSV peptide mass tag (2,074Da). Next, the three bead species were pooled in equal amounts and thepooled bead population was then deposited into the aforementionedpico-well plates by brief centrifugation. MALDI-TOF imaging (scanning)of the pico-well plate was performed essentially as described in Example2, in order to detect the HSV peptide mass tags from individual beads.

Verification of Bound Recombinant Proteins

In order to verify the presence of bound recombinant protein on thebeads, an aliquot of the same batch of beads was saved after loading therecombinant proteins but before loading the HSV peptide mass tags. Thesebeads were probed with a fluorescently (Cy3) labeled anti-VSV-G tagantibody (Sigma-Aldrich, St. Louis, Mo.) in order to detect this commonN-terminal epitope tag present in all the nascent recombinant proteins.To do so, beads were probed with 200 μL of antibody diluted from themanufacturer supplied stock at 1/50 in Block Buffer. Probing wasperformed for 30 min with mixing. Using the aforementioned FiltrationDevices, beads were then washed 4×400 μL briefly with TBS-T and then2×400 μL with TBS.

Beads were then embedded in a thin polyacrylamide film on a glassmicroscope slide for fluorescence imaging. The acrylamide mix wasprepared by mixing 487 μL TBS, 113 μL of a 40% acrylamide andbis-acrylamide mixture (19:1 ratio; Bio-Rad Laboratories, Hercules,Calif.), 1 μL, of 100% TEMED (Bio-Rad Laboratories, Hercules, Calif.)and 6 μL of a 10% (w/v) ammonium persulfate solution (prepared inwater). This acrylamide mix was used to resuspend the washed bead pelletto form 1% (v/v) beads. Approximately 10-20 μL of the bead suspensionwas placed on a standard glass microscope slide, overlaid with an 18 mmround microscope cover glass and allowed to polymerize for approximately10 min. The microscope slides were fluorescently imaged using a GenePix4200 laser based microarray scanner (Molecular Devices, Sunnyvale,Calif.).

Results:

Two recombinant human proteins (p53 and KLHL), each having a commonC-terminal HSV epitope tag, were expressed in a cell-free system andloaded/isolated onto anti-HSV antibody-coated 34 micron agarose beads.As a negative control, blank beads were also prepared in the same mannerexcept only the expressible DNA was omitted from the cell-free proteinsynthesis reaction. After protein expression and bead-capture, each ofthe three bead species was additionally loaded with a unique HSV peptidemass tag having a molecular weight of 1,368, 2,048 or 2,074 Da (bindingto beads again mediated by the anti-HSV antibody coating). The 3 beadspecies were then pooled and loaded into the aforementioned pico-wellplates. For MALDI-TOF mass spectrometry, the matrix was applied as athin and uniform film to the plate surface. The surface was then scannedin the MALDI-TOF mass spectrometry reflector mode in the 1,500-4,000 m/zspectral range. Three mass images were constructed using spectralintensity at the m/z corresponding to the molecular weight of the HSVpeptide mass tags. Three distinct populations of beads are visible onthe pico-well plate (FIG. 3A). The spectral analysis of each spotreveals a single strong MALDI-TOF peak, indicating that each beadcarries a homogenous population of peptide mass tag. Importantly,separate antibody-mediated fluorescence detection of the expressedrecombinant proteins on the beads demonstrates that in addition to themass tags, the expressed proteins are also present with signal-to-noiseratios of 10:1 and 60:1 for KLHL and p53 respectively (FIG. 3B). Thiswork verifies the ability of MALDI-TOF mass spectrometry to scan, imageand resolve individual beads and identify different analytes on thebeads (in this embodiment recombinant proteins) by virtue of co-loadedpeptide mass tags.

Example 4 Synchronization of Fluorescence Image and Mass SpectrometryMass-Image of Individually Resolved Beads

In this Example, 34 micron agarose beads coated with the anti-HSV tagantibody were loaded with cell-free expressed recombinant human p53 orrecombinant human KLHL12, both of which contained the HSV epitope tag;performed exactly as described in Example 3. Also as in Example 3, aparallel set of blank beads was prepared in the same manner except onlythe expression DNA was omitted from the cell-free reaction used tosynthesize the recombinant proteins.

Beads were then loaded with different HSV peptide mass tags labeled ontheir N-terminus with different fluorophores in order to create uniquemasses; done exactly as described in Example 3.

As a result of the previous steps in this Example, the three mass-taggedbead species were as follows: Blank beads (no recombinant protein) codedwith the unlabeled HSV peptide mass tag (1,368 Da), human p53 beadscoded with the Cy3 labeled HSV peptide mass tag (2,048 Da) and humanKLHL12 beads coded with the Cy5 labeled HSV peptide mass tag (2,074 Da).The three bead species were pooled, the pooled beads deposited in theaforementioned pico-well plates and the beads then mass-imaged withMALDI-TOF mass spectrometry exactly as described in Example 3, in orderto detect the mass tags on individual beads.

After MALDI-TOF mass-imaging, the same area of the pico-well plates wasfluorescently imaged using a GenePix 4200 laser based microarray scanner(Molecular Devices, Sunnyvale, Calif.).

In this Example, the mass-image of the Cy3 labeled HSV peptide mass tag(2,048 Da) was overlaid (synchronized) with the fluorescence (Cy3) imageof the same HSV peptide mass tag (same region of pico-well plate).Results in FIG. 4 show excellent concordance between the fluorescenceand mass images. The small number of discordant beads is believed to benon-specific autofluorescence of beads lacking the peptide mass tag,imperfections in the MALDI-TOF matrix coating and/or

Example 5 Photocleavable Mass Tags—Mass Spectrometry Readout andMass-Imaging from Individually Resolved Beads Preparation of NeutrAvidinCoated 34 Micron Agarose Affinity Beads

Performed in the same manner as in Example 3 for the anti-HSV antibodycoating of 34 micron agarose beads except in this case NeutrAvidinbiotin binding protein (Invitrogen, Carlsbad, Calif.) was conjugated tothe beads and loaded at a ratio 10 μg per μL of packed agarose beadvolume (NeutrAvidin concentration at binding step was 2.5 μg/μL).

Preparation of Photocleavable (PC) Biotin Labeled Peptide Mass Tags

Performed in the same manner as in Example 3 for the N-terminalfluorescence labeling of the HSV peptide mass tags except that thetarget peptide was the VSV-G peptide (YTDIEMNRLGK) (Roche AppliedScience, Indianapolis, 1N) (1,340 Da) and instead of using NHS-activated(primary amine reactive) fluorescence dye labeling reagents, AmberGen'sNHS-activated photocleavable (PC) biotin labeling reagent was used(AmberGen Incorporated, Watertown, Mass.) [Olejnik, Sonar,Krzymanska-Olejnik and Rothschild (1995) Proceedings of the NationalAcademy of Science (USA) 92: 7590-7594; Pandori, Hobson, Olejnik,Krzymanska-Olejnik, Rothschild, Palmer, Phillips and Sano (2002) ChemBiol 9: 567-73].

Binding of PC-Biotin Peptide Mass Tags to NeutrAvidin Agarose AffinityBeads

250 pmoles of the aforementioned PC-Biotin labeled VSV-G peptide masstag was added to 1.5 μL of packed NeutrAvidin agarose bead volume(45,000 beads) in 50 μL of Block Buffer (see Example 3 for buffercompositions). This corresponds to ˜5 (moles of PC-Biotin VSV-G peptidemass tag added per bead. The PC-Biotin VSV-G peptide mass tag wasallowed to bind for 30 min with mixing. Beads were then washed 3×400 μLbriefly with TBS-T, 3×400 μL TBS and then 4×400 μL with MassSpectrometry Grade Water (MSG-Water).

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Next, the beads loaded with the PC-Biotin VSV-G peptide mass tag werethen deposited into the aforementioned pico-well plates (Example 3) bybrief centrifugation. MALDI-TOF imaging (scanning) of the pico-wellplate was performed essentially as described in Example 2, in order todetect the VSV-G peptide mass tags from individual beads, with thefollowing exceptions: After bead deposition but before matrix coatingand MALDI-TOF imaging, photo-release of the captured mass tag wasachieved via illumination of the pico-well plates for 5 min with near-UVlight (365 nm peak UV lamp, Blak-Ray Lamp, Model XX-15, UVP, Upland,Calif.) at a 5 cm distance. The power output under these conditions wasapproximately 2.6 mW/cm² at 360 nm, to mW/cm² at 310 nm and 0.16 mW/cm²at 250 nm. For the minus light negative control (−UV), a portion of thepico-well plate was masked using an opaque solid barrier.

Results:

As demonstrated by the mass-image in FIG. 5, specific and robust VSV-Gpeptide mass tag signal is observed coming from individually resolvedbeads following light pre-treatment (+UV) to break the photocleavablebiotin linkage to the NeutrAvidin agarose beads prior to MALDI-TOF massspectrometry (MALDI-TOF mass spectrometer laser itself is inefficient atphotocleaving linker). Representative mass spectra are also shown fromindividual beads in FIG. 5. The VSV-G peptide is observed at its correctmass (1,340 Da), confirming full photocleavage of the PC-Biotin moietyfrom the peptide (which restores the peptide to its native unmodifiedstate). Very weak to no peptide signal is observed without lightpre-treatment (−UV) and no signal is observed when the peptide isomitted from the beads (−Mass Tag). Note that the saturating signalobserved in the light treated sample reduced the resolution of singlebeads by mass-imaging in this Example (although single beads are stillobserved). However, in the minus light permutation (−UV), thedramatically weaker signal better shows individually resolved 34 micronbeads by MALDI-TOF mass spectrometry mass-imaging in the pico-wellplates.

Example 6 Photocleavable Mass Tags (for Bead Identification) Co-Loadedwith “Bait” Molecules for Multiplex Bioassays: “Bait” Detection and MassSpectrometry Readout from Beads

One embodiment of mass spectrometry mass-imaging of beads or particlesis to load onto the beads both a mass tag for bead identification and“bait” molecules or compounds for use in multiplex bioassays. In thisExample, beads are co-loaded with both photocleavable (PC) peptide masstags for identification and human recombinant proteins as “bait”compounds.

Preparation of Dual Affinity Beads Coated with Both NeutrAvidin and theAnti-HSV Tag Capture Antibody

Performed in the same manner as in Example 3 for the anti-HSV antibodycoating of 34 micron agarose beads except in this case both the anti-HSVantibody and NeutrAvidin (Invitrogen, Carlsbad, Calif.) were conjugatedto the same batch of beads. In this case, instead of 6 μg of anti-HSVantibody per μL packed agarose bead volume as done in Example 3, 4 μg ofanti-HSV antibody and 2 μg of NeutrAvidin (6 μg total protein) wasco-loaded per each μL, of packed agarose bead volume.

Fluorescent Labeling of Dual Affinity Beads for Bead-ELISA Assay

The aforementioned dual affinity beads were directly labeled withfluorescence in order to enable normalization of total bead number persample in downstream bead-ELISA assays (see later in this Example forBead-ELISA). The beads were fluorescently labeled as follows: Theaforementioned Filtration Devices (see Example 3) were used tomanipulate the beads in the following procedures unless otherwise noted.Beads were washed 4× briefly with several bead bed volumes each ofConjugation Buffer (200 mM sodium bicarbonate and 200 mM NaCl). Beadswere then prepared to a 25% v/v bead suspension in Conjugation Buffer.Beads were then labeled with 270 pmoles of the Alexa Fluor® 594 SSElabeling reagent (Invitrogen, Carlsbad, Calif.) per each μL of packedbead volume (˜30,000 beads) for roughly 10 fmoles added labeling reagentper bead. Labeling reagent was added from a 27 mM stock in anhydrousDMSO. The labeling reaction was performed for 30 min with gentle mixingand protected from light. Beads were then washed 4× briefly in severalbead bed volumes of quench buffer (100 mM glycine in TBS; see Example 3for TBS) and then 2× briefly in several bead bed volumes of 0.1% BSA w/vin TBS. Beads were then prepared to a 10% v/v bead suspension in 0.1%BSA w/v in TBS and stored at +4° C. protected from light.

As a quality control measure, 1 μL of packed bead volume in 100 μL of0.1% BSA w/v in TBS was transferred to the wells of a 96-well opaqueblack flat bottom microliter plate. Beads were allowed to settle bygravity for 5 min and the Alexa Fluor® 594 fluorescence read in a TECANSpetraFluor Plus plate reader (Tecan Group Ltd., Männedorf, Switzerland)using a 560 nm excitation filter and 612 nm emissions filter. Usingseveral replicate samplings, fluorescence signal was compared to beadslacking the Alexa Fluor® 594 (same bead amount), yielding an averagesignal-to-noise ratio of 11:1.

Preparation of Photocleavable (PC) Biotin Labeled Peptide Mass Tags

Performed in the same mariner as in Example 5 except that the bradykininpeptide (Sigma-Aldrich, St. Louis, Mo.) (RPPGFSPFR) was used instead ofthe VSV-G peptide in Example 5.

Binding of PC-Biotin Peptide Mass Tags to Dual Affinity Beads

112.5 pmoles of the aforementioned PC-Biotin labeled bradykinin peptidemass tag was added to 0.75 μL of packed dual affinity agarose beadvolume (22,500 beads) in 225 μL of Block Buffer (see Example 3 forbuffer compositions). This corresponds to ˜5 fmoles of PC-Biotinbradykinin peptide mass tag per bead. The PC-Biotin bradykinin peptidemass tag was allowed to bind for 30 min with mixing. Beads were thenwashed 4×400 μL, briefly with TBS-T (see Example 3 for buffer). As anegative control, a parallel batch of beads was processed in the samemanner except that the PC-Biotin labeled bradykinin peptide mass tag wasomitted.

Binding of Recombinant Protein as “Bait” to Dual Affinity Beads

Performed exactly as in Example 3 with the following exceptions: Theaforementioned dual affinity beads, with and without the PC-Biotinlabeled bradykinin peptide mass tag, were used for recombinant proteincapture instead of the anti-HSV beads from Example 3. To create beadscontaining the “bait” (nascent recombinant protein), the dual affinitybeads were loaded with cell-free expressed recombinant human p53 protein(see Example 3). The p53 contained the HSV epitope tag for binding tothe beads. Prior to capture on the beads, in this Example, the crudecell-free expression reactions was mixed with equal volume of 5% BSA w/vin TBS-T and pre-clarified by passing the solution through theaforementioned Filtration Devices (see Example 3). Also as in Example 3,a parallel set of blank beads was prepared in the same manner exceptonly the expression DNA was omitted from the cell-free reaction used tosynthesize the recombinant protein.

After loading the protein to the beads, washing was also performed as inExample 3. These bead samples (bead suspensions) were then each split,whereby half of each sample was used for a Bead-ELISA for detection ofthe bead-bound p53 bait molecules and the other half used for MALDI-TOFmass spectrometry for detection of the photocleavable peptide mass tag.Both procedures are detailed below.

Bead-ELISA for Detection of the Human Recombinant p53 “Bait” on theBeads

Beads were manipulated with the aforementioned Filtration Devices unlessotherwise noted. Beads were probed with 200 μL of a monoclonalanti-VSV-G horseradish peroxidase (HRP) conjugated antibody (Clone P5D4,Sigma-Aldrich, St. Louis, Mo.) to detect the bead-bound humanrecombinant p53 “bait” with contained this N-terminal epitope tag. Forprobing, the manufacturer supplied antibody (˜1 mg/mL) was diluted1/20,000 in Block Buffer (see Example 3 for buffer). Probing wasperformed for 30 min with gentle mixing. Beads were then washed briefly4×400 μL, with TBS-T and 2×400 μL with 0.1% BSA w/v in TBS. Each beadsample was then re-suspended in 100 μL of 0.1% BSA w/v in TBS andtransferred to the wells of a 96-well opaque black flat bottommicrotiter plate. Beads were allowed to settle by gravity for 5 min andthe Alexa Fluor® 594 fluorescence read (bead normalization signal) in aTECAN SpetraFluor Plus plate reader (Tecan Group Ltd., Männedorf,Switzerland) using a 560 nm excitation filter and 612 nm emissionsfilter.

Next, the anti-VSV-G HRP antibody was detected to measure the bead-bound“bait”, i.e. the recombinant human p53. Without further processing, toeach well of the microtiter plate containing the beads, 100 μL ofSuperSignal Pico Chemiluminescence ELISA substrate(Thermo-Fisher-Pierce, Rockford, Ill.) was added and mixed for 15 min ona plate shaker. Beads were again allowed to settle by gravity for 5 minand the signal read in the TECAN SpetraFluor Plus plate reader (TecanGroup Ltd., Männedorf, Switzerland) using the instrument's luminescencemode.

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Analysis fromBeads

To detect the bead-bound PC-Biotin bradykinin peptide mass tag, analiquot of the same batch of beads that were loaded with the mass tag aswell as the recombinant human p53 (see earlier in this Example) wasanalyzed by mass spectrometry. This aliquot of beads was further washed3×400 μL briefly with TBS-T, 3×400 μL TBS and then 4×400 μL with MassSpectrometry Grade Water (MSG-Water) using the aforementioned FiltrationDevices. The beads were suspended in a small volume of MSG-Water andphoto-release of the bead-bound mass tag was achieved via illuminationfor 5 min with near-UV light (365 nm peak UV lamp, Blak-Ray Lamp, ModelXX-15, UVP, Upland, Calif.) at a 5 cm distance. The power output underthese conditions was approximately 2.6 mW/cm² at 360 nm, 1.0 mW/cm² at310 nm and 0.16 mW/cm² at 250 nm. The supernatant was then mixed withthe MALDI-TOF matrix and analyzed under standard conditions (note thatsingle bead mass-imaging was not done here).

Results:

FIG. 6A shows the results of the Bead-ELISA used to detect the presenceof the “bait” compound on the beads. In this case, human recombinant p53protein was the “bait” and was detected by an HRP labeled antibodydirected against its N-terminal VSV-G epitope tag. The Bead-ELISA signalreadout was from an entire population of beads by way of enzymaticchemiluminescence signal generation. To normalize for the amount ofbeads present in each sample (due to bead pipetting variance), the beadsalso carried a directly-conjugated fluorescence label, which was alsoread from the entire bead population. The p53 signal (i.e.chemiluminescent anti-VSV HRP signal) in FIG. 6A was normalized to thefluorescence signal (relative bead amount). Results show the p53 wasdetected both with and without the PC-Biotin bradykinin peptide mass tagpresent on the beads. In this Example, the presence of the mass tag doesreduce the binding capacity of the beads for p53 protein byapproximately 50%. No p53 signal was observed when the p53 was absentfrom the beads, whether or not the mass tag was present. Thesignal-to-noise ratio for the p53 signal was 42:1 for the mass-taggedbeads and 82:1 for the non mass-tagged beads.

An aliquot of the same batch of beads containing the bound PC-Biotinbradykinin peptide mass tag and bound recombinant p53 “bait” protein wasalso analyzed by MALDI-TOF mass spectrometry, following photocleavage ofthe mass tag from the beads. In this Example, the photo-released masstag from an entire population of beads was measured in bulk byconventional MALDI-TOF mass spectrometry, but other embodimentsenvisioned would involve mass-imaging of individually resolved beadssimilar to as done in Examples 5 and 7. Results in FIG. 6B show that thephotocleaved bradykinin peptide mass tag was observed at its correctmass (1,060 Da), and was detected on beads with or without bound p53. Nomass tag was observed on beads with our without the bound p53 in caseswhere the mass tag was not added to the beads.

Example 7 Photocleavable Mass-Tagged Probes for Mass SpectrometryReadout and Mass-Imaging from Individually Resolved Beads: AutoantibodyDetection in Autoimmune Disease

In this Example, specific probes were mass tagged with peptides and usedto detect analytes bound to “bait” molecules present on beads. Detectionwas by using MALDI-TOF mass spectrometry mass-imaging. Morespecifically, the “bait” molecule on the beads in this Example is arecombinant human protein, acting as an autoantigen (i.e. self antigenstargeted by serum autoantibodies in autoimmune disorders). The analytein this Example is an autoantibody (specific human IgG) present in theserum of a patient having an autoimmune disorder (Primary BiliaryCirrhosis or PBC in this case); whereby the autoantibody is detectedwith a mass-tagged anti-[human IgG] secondary antibody probe.

Binding of Recombinant Protein Autoantigen to Anti-HSV Agarose AffinityBeads

Performed exactly as in Example 3. First, to create beads containing the“bait” (nascent recombinant protein autoantigen in this Example), 34micron agarose beads coated with the anti-HSV tag antibody were loadedwith cell-free expressed recombinant human KLHL12 protein (PBCautoantigen; see US provisional filling at U.S. application No.61/248,768). The KLHL12 contained the HSV epitope tag for binding to thebeads. Also as in Example 3, a parallel set of blank beads was preparedin the same manner except only the expression DNA was omitted from thecell-free reaction used to synthesize the recombinant protein. Afterloading the protein to the beads, washing was also performed as inExample 3.

Preparation of the VSV-G PC-Biotin Labeled Peptide Mass Tag

Performed exactly as in Example 5.

Preparation of Fluorescent Tetrameric NeutrAvidin Protein as a Bridgefrom Probe to Mass Tag

A 5 mg/mL stock of tetrameric NeutrAvidin biotin binding protein(Invitrogen, Carlsbad, Calif.) was prepared in PBS (50 mM sodiumphosphate, pH 7.5, 100 mM NaCl). The stock was then mixed with equalvolume of 200 mM sodium bicarbonate 200 mM NaCl (no pH adjustment). 500μL of this solution was labeled with the Cy3-NHS ester reagent (GEHealthcare Life Sciences, Piscataway, N.J.) added from a 25 mM stock(stock in anhydrous DMSO) to yield a 10-fold molar excess of labelingreagent versus the NeutrAvidin. The reaction was carried out for 30 minwith gentle mixing and protected from light. Un-reacted labeling reagentwas removed by passing the solution through an Illustra NAP-5 G-25sepharose desalting column according to the manufacturer's instructions(GE Healthcare Life Sciences, Piscataway, N.J.) versus a TBS buffer.Concentration of the purified and labeled NeutrAvidin was determined bymeasuring absorbance at 280 nm.

Treatment of Autoantigen Beads with Human Autoimmune Serum andSubsequent Probing

The aforementioned beads, prepared with and without the KLHL12autoantigen and washed as described above, were then treated with eithera known KLHL12 autoantibody-positive PBC autoimmune serum or with aknown autoantibody-negative normal patient serum ProMedDx, LLC (Norton,Mass.). Both sera were previously confirmed KLHL12 autoantibody-positiveor negative by analysis on commercial human proteome microarraysperformed according the manufacturer's instructions (Human ProtoArray®4.0, Invitrogen, Carlsbad, Calif.). Serum treatment, probing and beadwashing steps were all performed in the aforementioned FiltrationDevices (see Example 3) unless otherwise noted. Sera were diluted1/1,000 in Block Buffer (see Example 3 for buffers unless otherwisenoted) and 200 μL was used to treat 1 μL packed bead volume (30,000beads) for each sample. Treatment was performed for 30 min with gentlemixing and the beads then washed 4×400 μL briefly with TBS-T. Beads werethen probed with 200 μL of a non-cleavable biotin labeled mouseanti-[Human IgG] secondary antibody (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) diluted to 10 μg/mL (˜65 nM) inBlock Buffer. Treatment was performed for 30 min with gentle mixing andthe beads then washed 4×400 μl briefly with TBS-T. Beads were thenprobed with 20 μL of the aforementioned Cy3 labeled NeutrAvidin dilutedto 4μg/mL (65 nM) in Block Buffer. Treatment was performed for 30 minwith gentle mixing and the beads then washed 4×400 μL briefly withTBS-T. Lastly, beads were then probed with 200 μL of the aforementionedPC-Biotin labeled VSV-G peptide mass tag diluted to 65 nM in BlockBuffer. Treatment was performed for 30 min with gentle mixing and thebeads were then washed 3×400 μL briefly with TBS-T, 3×400 μL TBS andthen 4×400 μL with Mass Spectrometry Grade Water (MSG-Water).

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Next, this same batch of beads was then split and deposited into twoseparate pico-well plates (see Example 3 for plates) by briefcentrifugation. One plate was used for MALDI-TOF mass spectrometrymass-imaging of individually resolved beads. Photocleavage of thePC-Biotin labeled VSV-G peptide mass tag from the beads in the platesand mass-imaging were performed as in Example 5, in order to detectbound autoantibody by virtue of the mass tag. The other plate was imagedfluorescently in a GenePix 4200 laser based microarray scanner(Molecular Devices, Sunnyvale, Calif.) to detect bound autoantibody byvirtue of the fluorescently labeled NeutrAvidin.

Results:

The top panel of FIG. 7 illustrates the design of the experiment. KLHL12(autoantigen) beads and blank beads (minus antigen) were prepared andprobed with the PBC autoimmune serum. A set of autoantigen beads wasalso probed with a normal serum as a negative control. The differentbead samples were kept separate in this Example. In this experiment,bead-bound autoantibody was probed with a biotinylated (non-cleavable)anti-[human IgG] secondary antibody, followed by tetrameric NeutrAvidinas a bridge, and finally, the photocleavable (PC) biotin labeled VSV-Gpeptide mass tag. To enable fluorescence detection of the bead-boundautoantibody, the NeutrAvidin bridge was labeled with Cy3.

The results in FIG. 7 (bottom panel) confirm that autoantibodies againstthe autoantigen can be detected both by fluorescence probing as well asMALDI-TOF mass spectrometry imaging of mass-tags from individuallyresolved beads, with high sensitivity and specificity. In both cases,strong positive signal is observed only on beads containing theautoantigen that were probed with the PBC autoimmune serum. Nosignificant signal is observed when autoantigen beads are probed with anormal serum or when blank beads are probed with the PBC autoimmuneserum. Fluorescence signal-to-noise ratio was 30 to 60:1, which is equalor greater than the highest signal-to-noise ratio observed on theaforementioned commercial ProtoArrays® for this serum-autoantigen pair.The MALDI-TOF mass spectrometry signal-to-noise ratio for the peak dueto the photocleaved PC-Biotin VSV-G peptide mass tag, in the case ofpositive sample, was 50:1 to 150:1, while for the two negative controls,the peak was indistinguishable from the noise (signal-to-noise ratio<2).

Example 8 Application of the Protease Enzyme to the Bead LibraryDeposited on the Pico-Well Plates: Efficient Protein Digestion withoutthe Loss of Spatial Resolution in MALDI-TOF Mass SpectrometryMass-Imaging

In this Example, a specific protein (recombinant human p53) bound to thebeads, which were deposited inside wells of a pico-well glass slide, wasdetected using both MALDI-TOF mass spectrometry imaging (MSI) andfluorescent scanning. The Example shows the ability to apply both theenzyme-containing solution and the MALDI matrix required for MALDIimaging in a manner that preserves the single-bead resolution of thearray.

Construction of the Probe Complex

The 34 micron agarose beads were coated with the anti-HSV tag antibody.Cell-free produced recombinant human p53 protein containing anN-terminal VSV tag and a C-terminal HSV tag was purified on beads asdescribed in Example 3. Subsequently, the protein was probed with abiotinylated anti-VSV antibody followed by incubation with a Cy3-labeledtetrameric NeutrAvidin and extensive wash to remove unbound fluorescentlabel.

Trypsin Digest of the Protein

The 34 micron beads containing the p53 probe complex were depositedinside wells of the pico-well glass slide. The bead density wasapproximately 1 bead per 20 wells. A dilute (25 μg/mL) aqueous solutionof mass-spectrometry grade trypsin was applied to the surface of theslide in the form of a fine mist using a Pari (Midlothian, Va.) LC®Sprint reusable nebulizer. Following trypsin application, the slide wasincubated for 1 hr at 37° C. to allow the digestion.

Application of the MALDI Matrix

For the purpose of MALDI imaging, the slides were coated with a thinlayer of α-hydroxy cinnamic acid (CHCA) MALDI matrix. A 16 mg/ml,solution of MS-grade CHCA in 60% of pure acetonitrile and 40% of 0.1%trifluoroacetic acid (v/v) was delivered to the surface of the slide inthe form of a fine mist using a Pari (Midlothian, Va.) LC® Sprintreusable nebulizer.

Fluorescence Scanning of the Bead Library following the TrypsinDigestion and MALDI Matrix Application

The slides were scanned using a GenePix 4200 laser based microarrayscanner (Molecular Devices, Sunnyvale, Calif.) at the 532 nm wavelengthcorresponding to the fluorescent signal of Cy3 (FIG. 8). First, theslides were scanned in the manufacturer's suggested orientation, withsignal detection from the bottom of the microwells through thefiberoptic channels. Afterward, the slides were turned upside down andthe same scan was performed with signal detection from the top of theslide. In the former configuration, the image reflects the analyte,which remains inside individual microwells since the scan is performedthrough individual channels. In the latter configuration, the imagereflects analyte located on the surface or near the surface of theslide, which was achieved by selecting the appropriate focus distance.Both images are very similar and show that the analyte, when measured byfluorescence, remains concentrated in the specific areas on the slidecorresponding to the locations of microwells. Note that the Cy3fluorescent label no longer remains bound to the protein-bead complexafter the trypsin digestion.

Mass-Spectrometry Readout of the Protein Digest

MALDI MSI scanning of the surface of the same fluorescently imagedpico-well slide showed a series of peaks in the 600-3,200 Da mass rangethat can be assigned to proteolytic fragments of p53 produced bydigestion of intact protein using trypsin. For example, the 890.4 Dapeak (FIG. 8, inset) corresponds to the fragment TEEENLR (amino acids308-314 in the recombinant protein sequence). The data shows that theprotein bound to the beads, which are deposited into the microwells, isefficiently digested and can be analyzed by MALDI mass spectrometry.

The above results demonstrate that digestion of protein on individualbeads followed by application of MALDI matrix does not decrease theresolution of the bead array and individual beads can still be resolvedeven at 10 micron resolution of the fluorescence scanner. Moregenerally, it is expected that application of other enzymes or compoundsdissolved in either aqueous or organic solution to the bead array can beperformed in a manner that preserves the resolution of the bead array.

Example 9 Measurement of Changes in the Protein Concentration Using aCombination of Protein Isotope Labeling, Proteolytic Digestion andMALDI-TOF Mass Spectrometry Mass-Imaging Analysis of Bead Microarrays

This Example shows the ability to measure changes in the concentrationof a specific protein (recombinant p53) obtained from two differentsources using MALDI bead microarrays. This is useful, for example, whenchanges in the protein expression between two different cell types needto be measured for multiple proteins. The approach involves: (1)expressing proteins separately in the non-labeled and isotope-labeledmedium that result in incorporation of the isotope label into thesynthesized proteins; (2) combining the two samples and purifying theaforementioned proteins on affinity beads such as antibody beads; (3)arranging beads into the microarray; (4) performing on-bead proteolyticdigestion and (5) measuring ratio of non-labeled versus isotope-labeledproteolytic fragments, which is indicative of the ratio of proteins inthe starting mixture.

Protein Isotope Labeling

Recombinant human p53 was expressed in a cell-free translation reactionsupplemented with non-labeled (natural abundance) amino acid mix.Separately, p53 was expressed in a reaction supplemented with ¹³C₆-Leuamino acid mix. Incorporation of an isotope labeled Leucine into theprotein chain results in a mass shift of +6 Da per each Leucine residue.

Affinity Purification

After the cell-free translation, the protein mixtures were separatelypurified on anti-HSV antibody-coated 34 micron agarose beads. In aseparate experiment, the protein mixtures were mixed in a 5:1 ratio(non-labeled vs labeled) before purification and subsequently purifiedon anti-HSV antibody-coated 34 micron agarose beads.

Trypsin Digestion

The bead mixtures were deposited on the MALDI plate and subject totrypsin digestion. In a separate experiment, the beads were depositedinto the pica-well glass slides and treated with trypsin.

MALDI-TOF Mass Spectra

The trypsin digest spectra acquired from beads, each carrying ahomogenous population of p53 protein (either labeled or non-labeled),reveal a series of peaks shifted by either +6 or +12 Da. For example(FIG. 9 A), the 1006 Da peak in the non-labeled protein is shifted to1018 Da in the labeled protein spectra. The mass-shifted peaks can beassigned to proteolytic segments of p53 containing one (+6 Da) or two(+12 Da) Leu residues.

Protein Quantification Using Isotope Labeling and MALDI MS Detection

Next, p53 was expressed in non-labeled and isotope labeled media and thetranslation reactions were mixed in a 5:1 ratio prior to binding to thebeads to mimic different levels of protein expression. Trypsin digestionand MS analysis were performed as described previously. The mass spectra(FIG. 9B) show that the ratio of 1006 to 1018 Da peaks is very close to5:1, matching the differences in protein amount.

Detection of Isotope-Shifted Peaks on Bead Microarrays

In this example, the mixture of beads carrying either pure non-labeledor pure isotope-labeled p53 proteins was deposited on the pico-wellslides, so that each well contains no more than one bead. The on-beadtrypsin digestion and MALDI matrix deposition were performed asdescribed in Example 8. The slide was scanned using MALDI MSI andsignals at 1,006 and 1,018 Da corresponding to the isotope-shifted p53proteolytic fragments were detected. As seen in FIG. 9C, two distinctnon-overlapping populations of beads were detected on the slide thatcorrespond to the two populations of p53 beads.

Example 10 Photocleavable DNA Tags and Bead Decoding by MassivelyParallel RT-PCR Chips

One embodiment of this invention involves use of PC-DNA tags to code anddecode beads. As an example, solid-phase (bead) PCR with universalphotocleavably attached primers, was used to separately amplify varioushuman open reading frame (ORF) plasmid inserts on a 34 micron agarosebeads; thus creating photocleavably tethered DNA amplicons (pure specieson each bead). Several different DNA-bead species were then pooled atvarious ratios and then photocleaved.

The photo-released DNA can be analyzed on a suitable instrument whichcan detect the DNA tags, such as a standard DNA hybridization chip (e.g.DNA microarray), a massively parallel DNA sequencer or an RT-PCR device.In the case of DNA hybridization chips, many chips are available such asfrom AffyMetrix (Santa Clara, Calif.) which have probes for thousands ofgenes that can be used to detect the release of specific DNA sequencesphoto-released from the beads. In this Example, a commercial prototypemassively parallel RT-PCR chip from WaferGen BioSystems Inc. (Fremont,Calif.) was used that can simultaneously analyze large numbers of suchPC-DNA tags. In this Example, WaferGen's 5,000-member prototype RT-PCRchip was used containing probes to all members of the test bead libraryevaluated. As shown in FIG. 10, gene ORFs were positively identifiedwhen photocleaved from as little as a single bead, with Cycle Threshold(Ct) values approximately following the bead numbers.

Example 11 Physical Pre-Selection of Beads for Decoding using aFluorescence Activated Cell-Sorting (FACS) Instrument

We evaluated in the feasibility of pre-isolating 34 micron agarose beadsusing fluorescence activated cell-sorting (FACS). Pre-isolation of onlythe positive beads of interest (e.g. by virtue of bound fluorescentprobes) greatly reduces the number of beads required to be decoded byMALDI-TOF mass spectrometry mass-imaging for example. Importantly, FACSis high throughput (can process millions of beads in a few minutes) andhas the ability for greater reproducibility and specificity thanmagnetic particle based affinity isolation methods, since beads can beanalyzed by multiple parameters on a bead-by-bead basis. In thisExample, blank protein beads and beads containing a recombinant proteinautoantigen for the autoimmune disease primary biliary cirrhosis (PBC)were separately prepared and probed with an appropriateautoantibody-positive human serum as detailed in Example 7. Boundautoantibody was detected with a fluorescently labeled secondaryanti-[human IgG] antibody (fluorescein). Beads were then analyzed usinga fluorescence activated cell sorting (FACS) instrument using acommercial service (BD FACS Vantage Cell Sorter; Cytometry Research LLC,San Diego, Calif.). As seen in FIG. 11, using the same cutoffs, 93% ofthe control beads (blank) were scored negative while 96% of theautoantigen beads were scored positive. Specificity of the fluorescencesignal is verified by analysis using a second fluorescence channel(Cy3), showing no significant signal.

Example 12 Photocleavable Mass Tags for Bead Identification and ProbeReadout in an Immune Response Profiling Scenario: MALDI-TOF Mass-Imagingof Individually Resolved Beads in an Array

One embodiment of mass spectrometry mass-imaging of beads or particlesis to load onto the beads both a mass tag for bead identification (“beadidentification tag”) and “bait” molecules or compounds for use inmultiplex bioassays. Furthermore, a probe (“prey”) used to treat (query)the beads can carry a different photocleavable mass tag for assayreadout (“probe tag”). In this scenario, mass-imaging of the beadsresults in two mass tag signals from those beads on which the bait hasbound its cognate probe, one mass tag for the bead identification tagand one for the probe tag. In this Example, human recombinant proteinsact as the “bait” compounds. An immune response profiling application isshown here as an example, whereby one of the bait proteins is a knownautoantigen and the other bait protein is a negative control(non-autoantigen). The beads are then treated with a human serum from anautoimmune patient known to have autoantibodies against saidautoantigen. To detect bound autoantibody, the beads are then probedwith an anti-[human IgG] secondary antibody which is ultimately detectedwith a unique photocleavable mass tag reporter (probe tag).

Preparation of Dual Affinity Beads Coated with Both NeutrAvidin and theAnti-HSV Tag Capture Antibody

Performed as in Example 6 (34 micron agarose beads).

Preparation of Photocleavable (PC) Biotin Labeled Peptide Mass Tags

Performed as in Example 5 except that the following peptides werelabeled: Bradykinin (RPPGFSPFR) (Sigma-Aldrich, St. Louis, Mo.) waslabeled for use as the probe tag and two custom peptides, obtainedcommercially from Sigma-Genosys (The Woodlands, Tex.), were labeled foruse as the bead identification tags (Tag-3.1=MIGGAGGRIR andTag-3.7=MIGGTGGRIR).

Binding of PC-Biotin Peptide Mass Tags to Dual Affinity Beads

Performed as in Example 6 except that 200 μL of PC-Biotin peptide masstag solution at a concentration of 0.75 pmoles/μL (150 pmoles) was addedto a 1 μL bead pellet volume (30,000 beads); for a final added amount ofPC-Biotin peptide mass tag of 5 fmoles per bead. As in Example 6, thisstep is to capture the PC-Biotin peptide mass tags on the dual affinitybeads by way of the NeutrAvidin coating on the beads (peptide captureefficiency not measured). In this Example, separate batches of beadswere prepared that were loaded with either the Tag-3.1 or the Tag-3.7mass tag, creating two pure populations of mass tag encoded beads. Aftercapture of the PC-Biotin peptide mass tags on beads and washing as inExample 6, the beads were quenched with 1 mM d-biotin in TBS-T (200 μL,per 1 μL bead pellet) (see Example 3 for buffer compositions). Quenchingwas performed for 30 min with mixing in the upper chamber of theFiltration Devices (see Example 3 for Filtration Devices and theirusage). The 1 μL bead pellets were then washed (in the FiltrationDevice) 4×400 μL briefly with TBS-T.

Binding of Recombinant Protein as “Bait” to Dual Affinity Beads

Performed essentially as in Example 6 with the following exceptions:Beads with the Tag-3.1 identification tag were loaded with cell-freeexpressed recombinant human Smith protein, a well known autoantigenbiomarker for systemic lupus erythematosus (SLE) [Mahler, Stinton andFritzler (2005) Clin Diagn Lab Immunol 12: 107-13]. Here, the β isoformof Smith was used (SmB). Beads with the Tag-3.7 identification tag wereloaded with cell-free expressed recombinant human GST A2 protein as anegative control (not a known autoantigen for SLE) (see Example 3 forcell-free protein expression). Treatment of the beads with the cell-freeexpression reactions for recombinant protein capture was performed as inExample 6, except that immediately following completion of theexpression reactions (prior to mixing with beads), protease inhibitorwas additionally added to the expression reactions to reduce thepossibility of subsequent proteolytic degradation of the peptide masstags on the beads (“Complete Mini Protease Inhibitor Cocktail Tablets”form Roche Applied Science, Indianapolis, Ind., Catalog Number11836153001; Stock Solution=1 mini-tablet in 1 mL of purified water; add1/10 volume of Stock Solution to completed cell-free protein expressionreactions). As in Example 6, the mechanism of capture of the cell-freeexpressed recombinant proteins on the dual affinity beads is by way ofthe anti-HSV antibody coating on the beads and the common C-terminal HSVepitope tag present in all expressed proteins.

Finally, after loading the recombinant proteins onto the beads, washingwas 4×400 μL briefly with TBS-T and 2×400 μL briefly with Block Buffer(see Example 3 for buffer compositions).

Immune Response Profiling Using the PC-Mass-Tagged and RecombinantProtein Loaded Beads

The two bead populations (separate), were each sequentially treated asfollows: All bead manipulations and washes were performed in theFiltration Devices unless otherwise noted (see Example 3 for theFiltration Devices and their usage). See Example 3 for buffercompositions. Beads (1 μL pellet volumes) were first probed with a knownSmB positive human serum from an SLE patient. Serum was diluted 1/1,000in 5% BSA (w/v) in TBS-T and 100 μL used to treat the beads for 30 minwith mixing. Beads were then washed 5×400 μL briefly with TBS-T. Beadswere then probed with 200 μL of a non-cleavable biotin labeled mouseanti-[Human IgG] secondary antibody (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) diluted to 10 μg/mL (˜65 nM) in 5%BSA/TBS-T supplemented with 1 mM d-biotin. Treatment was performed for30 min with gentle mixing and the beads then washed 4×400 μL briefly and2×400 μL for 5 min each with TBS-T. Beads were further washed 4×400 μLbriefly with 5% BSA/TBS-T. Beads were then probed with 200 μL of Cy3labeled NeutrAvidin (see Example 7) diluted to 4 μg/mL (˜65 nM) in 5%BSA/TBS-T. Treatment was performed for 30 min with gentle mixing and thebeads then washed 4×400 μL briefly with TBS-T. Lastly, beads were thenprobed with 200 μL of the aforementioned PC-Biotin labeled Bradykininpeptide mass tag (probe tag) diluted to 65 nM in 5% BSA/TBS-T. Treatmentwas performed for 30 min with gentle mixing and the beads were thenwashed 4×400 μL, briefly with TBS-T, 4×400 μL TBS and then 4×400 μL withMass Spectrometry Grade Water (MSG-Water).

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Performed in the pico-well plates as in Examples 5 & 7. Note that thetwo populations of bead species (SmB and GST) were pooled prior todeposition into the pico-well plates. In this Example, no florescenceimaging was used.

Confirmation of Mass-Imaging Results Using ELISA

Results of autoantibody detection on beads with the mass-imagingapproach were confirmed by testing the same SLE serum against the samecell-free expressed recombinant proteins using a 96-well microtiterplate based ELISA. For this, AmberGen's T²-ELISA™ was used (seeDescription of Invention for overview of T²-ELISA™). The procedures wereas follows:

Cell-Free Protein Expression for ELISA

See Example 3 for cell-free protein expression. Protein expressionreactions contained the cognate plasmid DNA while blank expressionreactions lacked only the plasmid DNA. Expression reactions were stoppedby diluting 1/20 in TDB [1% BSA (w/v) and 0.1% (v/v) Triton X-100 inTBS-T (50 mM Tris, pH 7.5, 200 mM NaCl, 0.05% (v/v) Tween-20)].

Enzyme-Linked Immunosorbent Assay (ELISA) for Autoantibody Detection

Nunc Brand 96-well Polysorp™ Microwell™ white opaque, flat bottom,untreated polystyrene microtiter plates (Nunc Brand from Thermo-FisherScientific, Rochester, N.Y.) were used for a sandwich type Enzyme-LinkedImmunosorbent Assay (ELISA). Plates were coated with 0.5 μg/mL of amouse monoclonal anti-HSV® tag capture antibody (EMD Biosciences, Inc.,San Diego, Calif.) in sodium carbonate/bicarbonate pH 9.3 for 30 minwith shaking (50 μL/well). Plates were then washed 6× in TBS-T (wellsfilled to maximum) on an ELx405 Select Robotic Plate Washer (BioTek,Winooski, Vt.). See Example 3 for TBS-T buffer composition. All platewashes were performed in this manner unless noted otherwise. Plates werethen blocked for 30 min at 300 μL/well in 1% BSA (w/v) in TBS-T. Thesolution was removed from the plates and the aforementioned stopped(i.e. diluted) cell-free expression reactions (protein and blankreactions) were then added at 100 μL/well and shaken for 30 min to allowthe nascent proteins to be captured by their common C-terminal HSVepitope tags. Plates were washed and the same SLE serum sample used forthe bead assays earlier in this Example (diluted at 1/1,000 in 1% BSA(w/v) in TBS-T) was added at 100 μL/well and shaken for 30 min. Theserum sample was run against wells of the proteins and wells of thecell-free expression blank. Additionally, one set of wells of proteinand one set of wells of the cell-free expression blank were designatedfor VSV-G epitope tag detection (common N-terminal tag in all expressedproteins), and therefore received plain 1% BSA (w/v) in TBS-T instead ofdiluted serum at this stage. To avoid contamination of the robotic platewasher with human serum, plates were subsequently washed 4× by manualaddition of TBS-T (wells filled to maximum) followed by vacuumaspiration and then washed 6× in the robotic plate washer as describedearlier in this Example. Wells designated for detection of the VSV-Gepitope tag then received an anti-VSV-G horseradish peroxidase (HRP)labeled monoclonal antibody (Clone P5D4, Roche Applied Science,Indianapolis, Ind.) diluted 1/20,000 in 1% BSA/TBS-T. Wells designatedfor detection of serum autoantibody received a mouse anti-[human IgG]HRP labeled monoclonal secondary antibody (minimum cross-reactivity withmouse immunoglobulin; Jackson ImmunoResearch Laboratories, Inc, WestGrove, Pa.) diluted 1/20,000 in 1% BSA/TBS-T. Plates were shaken for 30min. The solutions were then manually dumped from the plates byinversion followed by vigorous patting of the plates inverted on a drypaper towel to remove residual fluid. Plates were then washed in therobotic plate washer as described earlier in this Example.Chemiluminescence signal was generated by the addition of 50 mL/well ofSuperSignal ELISA Pico Chemiluminesence Substrate (Pierce Brand fromThermo Fisher Scientific, Rockford, Ill.). Plates were developed byshaking for 15 min and then read on a LumiCount luminescence platereader (1 s exposure, PMT of 650V, gain 1) (Packard/PerkinElmer Life andAnalytical Sciences, Inc., Boston, Mass.).

Results:

FIGS. 12A-C show the design and results of autoantibody profiling on thebeads using a fully PC-Mass-Tag based configuration. FIG. 12Aillustrates the basic experimental design. For this experiment, beadswere coated with both NeutrAvidin and the anti-HSV antibody. PC-Biotinlabeled peptide Mass-Tags (“Bead Identification Tags”) were used toencode the two bead populations and the unoccupied biotin binding siteson the NeutrAvidin were permanently quenched with soluble d-biotin(black circles in FIG. 12A). Beads were then loaded with cell-freeexpressed proteins (rabbit reticulocyte expression system). Either humanGST A2 (negative control) or SmB (SLE autoantigen) were loaded to thebeads by direct in situ capture through their common C-terminal HSV tag.Beads were then sequentially probed with a known SmB positive SLE serum,biotinylated anti-[Human IgG] secondary antibody, tetrameric NeutrAvidinas a bridge and a single species of PC-Biotin labeled Mass-Tag (“ProbeTag”). Note that although not done in this Example, in anotherembodiment, the NeutrAvidin bridge can be fluorescently labeled for bothfluorescence and mass-tag readout of bound autoantibody.

MALDI-TOF mass-imaging of the pooled beads following their depositioninto the pico-well plates was performed. Results in FIG. 12B (image 1)show excellent concordance between the Probe Tag (red) and only the SmBbeads as revealed by their Bead Identification Tag (green; offset inimage 1 of FIG. 12B). The negative control GST beads, revealed by theirspecific Bead Identification Tag (white/gray; overlaid in image 2 ofFIG. 12B), show no signal from the Probe-Tag as expected. In image 2 ofFIG. 12B, the SmB beads show as yellow spots in the direct image overlay(composite of red and green mass-tag signals from their Probe Tag andBead Identification Tag respectively).

To validate these results, we used a conventional 96-well microtiterplate ELISA assay (T²-ELISA™) formatted in analogy to the bead assay.Cell-free expressed proteins were immobilized on the ELISA well surfaceby anti-HSV antibody-mediated capture. Following serum treatment, wellswere probed with an enzyme-labeled (HRP) anti-[Human IgG] secondaryantibody to detect bound autoantibody. As an additional control, theamount of captured protein was detected in separate wells with areporter-labeled antibody to the common N-terminal VSV-G epitope tag inall expressed proteins. Results are shown in FIG. 12C. VSV-G epitope tagsignal reveals that both GST and SmB were expressed and captured atsimilar levels, while the expression blank shows no significant signal(expression reaction without DNA). However, the autoantibody readoutshows the SLE serum (same as in bead assay) reacts only with SmB asexpected.

Example 13 MALDI-TOF Mass-Imaging of 10 Unique Photocleavable Mass-TagEncoded Bead Species in an Array: Mass-Imaging for Identification inConjunction with Antibody Detection of a Bead-Bound Bait Protein

In this Example, 10 distinct species of beads, each carrying uniquephotocleavable peptide mass tags, and 1 additionally containing arecombinant protein, were prepared and arrayed in the pico-well plates.The array of all 10 bead species, randomly distributed, was thenmass-imaged using MALDI-TOF.

All beads in this Example carried both a unique peptide mass tag foridentification as well as a common capture antibody. The antibody servesto bind recombinant proteins that contain a common epitope tag. Thecaptured recombinant proteins act as “bait” for specific probes(“prey”), such as a fluorescently labeled detector antibody. In thisExample, 1 of the 10 bead species carried cell-free expressedrecombinant p53 protein (captured by antibody) in addition to thepeptide mass tag. The p53 beads were additionally detected using afluorescent antibody directed against an epitope tag in the p53 protein.

Preparation of Dual Affinity Beads Coated with Both NeutrAvidin and theAnti-HSV Tag Capture Antibody

Performed as in Example 6 (34 micron agarose beads).

Preparation of Photocleavable (PC) Biotin Labeled Peptide Mass Tags

Performed as in Example 5 except that the following custom peptidesobtained commercially from Sigma-Genosys (The Woodlands, Tex.) were usedfor labeling (peptide sequence in brackets):

 1. Tag-1.1 [QRPDVTR]  2. Tag-2.3 [DIEHNR]  3. Tag-2.8 [DIERNR]  4.Tag-3.1 [MIGGAGGRIR]  5. Tag-3.2 [MIGGEGGRIR]  6. Tag-3.4 [MIGGIGGRIR] 7. Tag-3.5 [MIGGSGGRIR]  8. Tag-3.6 [MIGGPGGRIR]  9.Tag-3.7 [MIGGTGGRIR] 10.   Tag-3.8 [MIGGRGGRIR]

Binding of PC-Biotin Peptide Mass Tags to Dual Affinity Beads

Performed as in Example 6 except that 200 μL of PC-Biotin peptide masstag solution at a concentration of 0.75 pmoles/μL (150 pmoles) was addedto a 1μL bead pellet volume (30,000 beads); for a final added amount ofPC-Biotin peptide mass tag of 5 fmoles per bead. As in Example 6, thisstep is to capture the PC-Biotin peptide mass tags on the dual affinitybeads by way of the NeutrAvidin coating on the beads (peptide captureefficiency not measured). In this Example, 10 separate batches of beadswere prepared, each batch uniquely loaded with 1 of the 10aforementioned peptides, creating 10 pure populations of unique mass tagencoded beads. After capture of the PC-Biotin peptide mass tags on beadsand washing as in Example 6, the beads were quenched with 1 mM d-biotinin TBS-T (200 μL per 1 μL bead pellet) (see Example 3 for buffercompositions). Quenching was performed for 30 min with mixing in theupper chamber of the Filtration Devices (see Example 3 for FiltrationDevices and their usage). The 1μL bead pellets were then washed (in theFiltration Device) 4×400 μL briefly with TBS-T.

Binding of Recombinant Protein as “Bait” to Dual Affinity Beads

Although in some embodiments all mass tagged bead species canadditionally carry a “bait” molecule such as a protein, in this Example,with the exception of the Tag-3.7 beads, the other 9 mass tagged beadspecies were not loaded with cell-free expressed recombinant protein andthus not subjected to this portion of the procedure:

Beads with the Tag-3.7 identification tag were loaded with cell-freeexpressed recombinant human p53 protein essentially as in Example 6 withthe following exceptions: Treatment of the beads with the cell-freeexpression reaction for recombinant p53 protein capture was performed asin Example 6, except that immediately following completion of theexpression reaction (prior to mixing with beads), protease inhibitor wasadditionally added to the expression reaction to reduce the possibilityof subsequent proteolytic degradation of the peptide mass tags on thebeads (“Complete Mini Protease Inhibitor Cocktail Tablets” form RocheApplied Science, Indianapolis, Ind., Catalog Number 11836153001; StockSolution=1 mini-tablet in 1 mL of purified water; add 1/10 volume ofStock Solution to completed cell-free protein expression reactions). Asin Example 6, the mechanism of capture of the cell-free expressedrecombinant p53 protein on the dual affinity beads is by way of theanti-HSV antibody coating on the beads and the C-terminal HSV epitopetag present in the recombinant p53. Finally, after loading therecombinant p53 protein onto the Tag-3.7 encoded beads, washing was4×400 μL briefly with TBS-T and 2×400 μL briefly with Block Buffer (seeExample 3 for buffer compositions).

As a negative control, a separate “blank” bead sample was prepared,corresponding to beads treated with a cell-free expression reaction thatwas performed lacking only the p53 DNA.

Pooling Beads and Fluorescence Detection of p53 Beads

All 10 bead species were then pooled in equal numbers. As a control, analiquot of the pure p53 beads was also set aside. This created 3 beadpopulations (“samples”): The 10-species pool, the p53 beads and theblank beads (see above). Each of the 3 bead samples was then probed witha fluorescently labeled anti-VSV-Cy3 antibody to specifically detect thep53 beads, by way of the VSV epitope tag present in the recombinant p53.After fluorescence probing, an aliquot of the 10-species pooled samplewas set aside for MALDI-TOF mass-imaging (next section). All remainingportions of the 3 bead samples were embedded in a polyacrylamide film ona microscope slide and fluorescently imaged in a microarray scanner. Thefluorescence antibody probing, embedding and fluorescence imaging wasperformed as in Example 3 in the section headed “Verification of BoundRecombinant Proteins”.

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Performed in the pico-well plates as in Examples 5 & 7.

Results:

This Example successfully shows the ability to mass-image 10 distinctpeptide-bead species (mass-tagged beads), using 34 micron beads arrayedin the pico-well plates (peptides affinity captured on beads with aphotocleavable linker). FIG. 13A shows an overlaid mass-image, wherebyall signals from all 10 peptide mass tag species are colored red (pixelintensity proportional to mass spectral intensity). Regions of overlapof any two or more peptide mass tag species are colored yellow. As seen,individual 34 micron beads can be imaged by virtue of the mass of theunique peptides originating from them. Negligible overlap betweendifferent peptide species in the mass image is observed (<5 beads infield of view), thus confirming the spatial resolution of single beadsin the mass image. The panels in FIG. 13B show separate mass images foreach the 10 peptide-bead species for the same region (grayscale only).As seen, beads corresponding to all 10 peptide mass tag species areequally represented in the mass-image.

To confirm that peptide mass tag imaging of beads is compatible with thepresence of “bait” molecules on the same beads, such as a recombinantprotein, an aliquot of the same beads was subjected to fluorescenceimaging on a microscope slide. Note: These beads had also been probedwith a florescent antibody directed specifically against an epitope tagin the p53 protein. Results are shown in FIG. 13C. As can be seen,control beads corresponding to the pure population of p53 beads beforepooling as well as the negative control (blank) beads, show that a p53is specifically detected by fluorescence using a specific antibody probe(“prey”). In the “Higher Contrast” image, blank beads are slightlyvisible via their non-specific background fluorescence. Signal-to-noisefor p53 detection was ˜25:1. In the image of the pooled beads, p53 beadsare distinguished by their strong fluorescence, while the othermass-tagged non-p53 beads are slightly visible again via theirnon-specific background fluorescence.

Example 14 Direct Chemical Linkage of Photocleavable Mass Tags to BeadSurfaces: Elimination of the Need for Affinity Based Linkages

The previous Examples used affinity linkages to attach peptide mass tagsto beads for MALDI-TOF mass-imaging and decoding, e.g. bead-boundantibodies used to capture epitope-containing peptide mass tags inExample 3, or bead-bound (strept)avidin used to capture photocleavablebiotin labeled peptide mass tags in Example 13. However it is possible,and in fact desirable in some cases, to directly attach mass tags(peptides or otherwise) to beads using a direct chemical linker by wayof a covalent bond, whereby the linker is photocleavable. Thiseliminates the need for an affinity capture agent (e.g. antibody or(strept)avidin)), which may interfere with some downstream applications.This Example will show one embodiment of this:

In this Example, the compound shown in FIG. 14A (upper left panel) willbe synthesized (“Photocleavable Amine Linker”). This compound willconsist of a protected amine moiety on one end, an activatedamine-reactive NHS ester on the other end and a spacer arm andphotocleavable nucleus in between. As shown in FIG. 14A, the NHS esterwill be for attachment of the compound to an amine-bearing mass tag(e.g. peptide) and the protected amine in the compound will be for laterattachment, following de-protection, of the modified mass tag to asurface, such as a bead, or to another molecule such as a probe(“prey”), for example an antibody probe. It is understood that this“Photocleavable Amine Linker” is one example compound of many possiblecompounds that can be used for direct covalent attachment ofphotocleavable mass tags to surfaces or to other molecules (e.g.probes). For instance, instead of an activated NHS ester, otheractivated or react-able moieties can be used, such assulfhydryl-reactive maleimide moieties, or carboxyl or phosphatemoieties which can be cross-linked to mass tags using carbodiimidechemistries. Likewise, the protected amine can be replaced by adifferent moiety (protected if necessary) to facilitate attachment ofthe modified photocleavable mass tag to a surface (e.g. bead) or toanother molecule (e.g. probe). Finally, the while a hydrocarbon spacerarm is shown in the current compound, other spacers are possible, forexample a 2,2′-(ethylenedioxy)-bis-(ethylamine) spacer could be used forbetter solubility in an aqueous environment [Pandori, Hobson, Olejnik,Krzymanska-Olejnik, Rothschild, Palmer, Phillips and Sano (2002) ChemBiol 9: 567-73].

In this Example, FIGS. 14A and B show one embodiment to be tested thatwill use the “Photocleavable Amine Linker” to modify a peptide mass tag.Following mass tag modification, the amine on the “Photocleavable AmineLinker” will be de-protected. The protecting group on the amine can beacid labile, such as a Boc protecting group, or base labile, forinstance an Fmoc protecting group (or a protecting group cleavable byother means). In this case, a Boc protecting group will be used andtrifluoroacetic acid (TEA) will be used for de-protection. Followingde-protection, the modified peptide mass tag will be purified using agel filtration column.

Following preparation of the photocleavable mass tags as describedabove, experiments will be performed similar to Example 13, except thatinstead of attachment of photocleavable biotin labeled peptide mass tagsto NeutrAvidin coated beads, the photocleavable amine modified peptidemass tags synthesized here will be directly attached to theNHS-activated beads (FIG. 14B). This mass tag attachment will be donesimultaneously with the attachment of the anti-HSV capture antibody tothe NHS-activated beads, under the same buffer conditions (see Examples6 & 13 for beads and anti-HSV antibody attachment). In this Example,unlike Example 13, there will be no NeutrAvidin or (strept)avidinattached to the beads, as it will not be needed. In this Example, uponillumination with the proper light, the directly and covalently attachedmass tags will be photo-released from the beads (FIG. 14B). In this way,MALDI-TOF mass-imaging of mass tag encoded beads arrayed in thepico-well plates will be possible, analogous to experiments in Example13.

One expected benefit will be less background noise in assay readout(e.g. probe detection) and generally less interference with downstreamassays due to the lack of a NeutrAvidin or (strept)avidin coating on thebeads (e.g. less interference with assays involving in situ, on-beadprotease digestion of bead-bound proteins such as in Example 9).

In a related embodiment that will be evaluated, a compound similar tothe “Photocleavable Amine Linker” shown in the upper left panel of FIG.14A will be synthesized, except that the protected amine group in the(green and red boxes) will be replaced by a sulfhydryl-reactivemaleimide group, to create a bi-functional photocleavable cross-linker.In this scenario, the NHS reactive portion of the compound will still beused to modify a mass-tag (e.g. peptide), such as discussed above inthis Example and shown in FIG. 14A, however, instead of subsequentlyattaching the modified mass-tag to a bead surface (although stillpossible with this compound), it will subsequently be attached to aprobe (“prey”), such as an antibody, which contains native or addedsulfhydryl groups for modification. In this manner, the probe, whichwill now bear a photocleavable mass-tag, can be used query beads forexample, such as done in Examples 7 and 12, to allow readout of theprobe binding by MALDI-TOF mass-imaging of the beads. This will allowmass-tag coding of different probe (“prey”) molecules, as well asmass-tag coding of entire bio-samples or heterogeneous mixtures (e.g. ablood serum sample), to facilitate multiplexing at the level of theprobes or samples used to query beads.

Example 15 Mass Spectrometry Readout and Mass-Imaging from IndividuallyResolved 34 Micron Beads in Metal Coated Pico-Well Plates

In this Example, fluorescence imaging and MALDI-TOF mass-imaging ofbeads in plain and gold-coated pico-well plates was evaluated.Conductive surfaces are typically more ideal for MALDI-TOF as they avoidcharge buildup.

Pico-Well Plates

Pico-well plates (Incom Inc., Charlton, Mass.) as described in Example 3were either used as is, or coated with a thin layer of gold by themanufacturer (Incom Inc., Charlton, Mass.). Briefly, coating involvesplasma cleaning of the plates, then sputtering on a thin coat oftitanium to promote adhesion of gold to the glass, and then applying a 5nm layer of gold on top of that. SEM (scanning electron microscopy), EDX(energy dispersive X-ray analysis) and AFM (atomic force microscopy)were used by the manufacturer to verify uniform coating of the plates.

Preparation of Dual Affinity Beads Coated with Both NeutrAvidin and theAnti-HSV Tag Capture Antibody

Performed as in Example 6

Preparation of Photocleavable (PC) Biotin Labeled Peptide Mass Tags

Performed in the same manner as in Example 5 except that the bradykininpeptide (Sigma-Aldrich, St. Louis, Mo.) (RPPGFSPFR) was used instead ofthe VSV-G peptide in Example 5.

Binding of PC-Biotin Peptide Mass Tags to Dual Affinity Beads

Performed as in Example 6 except that 200 μL of PC-Biotin peptide masstag solution at a concentration of 0.75 pmoles/μL (150 pmoles) was addedto a 1 μL bead pellet volume (30,000 beads); for a final added amount ofPC-Biotin peptide mass tag of 5 fmoles per bead. As in Example 6, thisstep is to capture the PC-Biotin peptide mass tags on the dual affinitybeads by way of the NeutrAvidin coating on the beads (peptide captureefficiency not measured).

Binding of Recombinant Protein as “Bait” to Dual Affinity Beads

Beads with the bradykinin mass-tag were loaded with cell-free expressedrecombinant human p53 protein essentially as in Example 6 with thefollowing exceptions: Treatment of the beads with the cell-freeexpression reaction for recombinant p53 protein capture was performed asin Example 6, except that immediately following completion of theexpression reaction (prior to mixing with beads), protease inhibitor wasadditionally added to the expression reaction to reduce the possibilityof subsequent proteolytic degradation of the peptide mass tags on thebeads (“Complete Mini Protease Inhibitor Cocktail Tablets” form RocheApplied Science, Indianapolis, Ind., Catalog Number 11836153001; StockSolution=1 mini-tablet in 1 mL of purified water; add 1/10 volume ofStock Solution to completed cell-free protein expression reactions). Asin Example 6, the mechanism of capture of the cell-free expressedrecombinant p53 protein on the dual affinity beads is by way of theanti-HSV antibody coating on the beads and the C-terminal HSV epitopetag present in the recombinant p53. Finally, after loading therecombinant p53 protein onto the bradykinin encoded beads, washing was4×400 μL briefly with TBS-T and 2×400 μL briefly with Block Buffer (seeExample 3 for buffer compositions).

Pooling Beads and Fluorescence Detection of p53 Beads

Beads were then probed with a fluorescently labeled anti-VSV-Cy3antibody to specifically detect the p53 beads, by way of the VSV epitopetag present in the recombinant p53. The fluorescence antibody probingwas performed as in Example 3 in the section headed “Verification ofBound Recombinant Proteins”. For both the plain glass and gold-coatedpico-well plates, imaging of fluorescence was performed through thebottom of the plates (i.e. thorough the fiber optics).

In Situ Trypsinization for Gold Versus Glass Comparison

In a second experiment, beads were created carrying recombinant proteinscaptured via the anti-HSV antibody similar to as earlier in thisExample, but without any mass-tags and without any antibody probingsteps. Beads were deposited into gold-coated and glass pico-well platesand in situ trypsin digestion was performed similar to as in Example 8.In this example human ACVR-2B was used as the recombinant protein.

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Performed in the plain glass and gold-coated pico-well plates as inExamples 5 & 7. Photocleavage was not used for in situ trypsin digestedsamples.

Results:

Results in FIG. 15A show MALDI-TOF mass-imaging of the photocleavablebradykinin mass-tag and fluorescence imaging of the anti-p53 antibodyprobe from the 34 micron beads in both the gold-coated and plain glasspico-well plates. Note that the fluorescence and MALDI-TOF images are ofdifferent regions of the plate in this Example. The data show thatindividually resolved beads can be imaged both by MALDI-TOF and byfluorescence in both the gold-coated and plain glass pico-well plates.Note that the contrast settings of the fluorescence images in FIG. 15Afor the gold-coated and plain glass pico-well plates are not the sameand thus cannot be directly compared in FIG. 15A. However, based on theactual raw fluorescence intensity values, the gold-coated platesproduced a weaker fluorescence signal by approximately 2-3 fold.

Results in FIG. 15B show direct comparison of MALDI-TOF spectra frombeads in both the gold-coated and plain glass pico-well plates followingin situ trypsin digestion of bead-bound human recombinant ACVR-2B.Various peptide peaks are visible in both types of pico-well plates,arising from proteolytic fragments of the ACVR-2B and capture antibody,as well as non-specifically bound peptides (e.g. from the expressionlysate). However, the gold-coated plates produce more detectiblefragments and an overall signal increase of roughly 2-fold (see “RawIntensity Max” scale on the right-hand y-axis for quantitativecomparison). Furthermore, the gold-coated plates allow a greater rangeof mass coverage. While the plain glass pico-well plates show virtuallyno detectible peptides above 1,650 Da (see green markings in FIG. 15B),the gold-coated plates show a multitude of peptides >1,650 Da. Finally,in this Example, only the gold-coated plates yield peptide fragmentswhich can be assigned to ACBR-2B protein sequence based on their mass(see red labels in FIG. 15B).

Example 16 MALDI-TOF Mass-Imaging of Multiple Unique PhotocleavableMass-Tag Encoded Bead Species in an Array: Synchronizing Mass-Image forBead Identification with Fluorescence Antibody Detection of a Bead-BoundBait Protein

In this Example, 2 distinct species of beads, each carrying a uniquephotocleavable (PC) peptide mass tag, and 1 of the 2 additionallycarrying bound recombinant p53 protein (“bait”), were preparedseparately, pooled and then probed with a fluorescently labeled antibody(“prey”) to detect the recombinant p53 protein. A 3rd bead species wasthen spiked in as “Marker Beads”. These Marker Beads carried a uniquephotocleavable peptide mass tag for identification and the bead surfacewas covalently labeled with a fluorophore having different anddistinguishable spectral properties than that used on the antibodyprobe. The pool of 3 bead species was randomly arrayed in the pico-wellplates and then imaged by mass and by fluorescence.

Preparation of NeutrAvidin Coated Beads

NeutrAvidin coated 34 micron agarose beads were prepared as in Example5. As described below, these NeutrAvidin beads were used to prepare boththe “Dual Affinity Beads” and the “Marker Beads”.

Preparation of Dual Affinity Beads Coated with NeutrAvidin Directly andIndirectly with the Anti-HSV Tag Capture Antibody

The aforementioned NeutrAvidin beads were then loaded a biotin labeledanti-HSV tag polyclonal antibody as follows: Goat anti-HSV tagpolyclonal antibody was purchased from Bethyl Laboratories (Montgomery,Tex.), provided at 1 mg/mL in PBS. 800 μL of this antibody solution (800μg) was then mixed with 1/9^(th) volume of 1M sodium bicarbonate. Theantibody was biotin labeled by adding a 10-fold molar excess ofEZ-Link-Sulfo-NHS-LC-Biotin (Thermo-Fisher-Pierce, Rockford, Ill.) andreacting for 30 min with gentle mixing. The reaction was quenched byadding 1/9^(th) volume of 1M glycine for 15 min with gentle mixing. Thelabeled antibody was then purified on a PD MidiTrap G-25 desaltingcolumn against TBS (see Example 3 for buffer) and according to themanufacturer's instructions (GE Healthcare Life Sciences, Piscataway,N.J.). The purified and biotin-labeled antibody was then diluted to 0.15μg/μL in TBS-T (see Example 3 for buffer). Using this solution, theNeutrAvidin beads were coated at a ratio of 12 μg of the biotin labeledanti-HSV tag antibody per each 1 μL of actual bead pellet volume for 30min with gentle mixing. Beads were then washed 4× briefly with an excessof TBS-T using the aforementioned Filtration Devices (see Example 3 fordevices). Beads were stored as a 20% (v/v) suspension at +4° C.

Note that because the antibody does not saturate all the biotin bindingsites on the NeutrAvidin beads, it was therefore possible toadditionally load PC-Biotin labeled peptide mass tags onto the beads(see later steps in this Example).

Preparation of “Marker Beads” Coated with NeutrAvidin Directly andLabeled with Fluorescence

Performed as in Example 6 except that the aforementioned NeutrAvidinbeads (no bound antibody) were used for fluorescence labeling and theCy5-NHS activated (primary amine reactive) fluorescent dye labelingreagent was used (GE Healthcare Life Sciences, Piscataway, N.J.).

Preparation of Photocleavable (PC) Biotin Labeled Peptide Mass Tags

Performed as in Example 5 except that the following commerciallyavailable peptides were used for labeling with PC-Biotin (peptidesequence in brackets) (all peptides purchased for labeling were fromAnaSpec, Fremont, Calif., except for Bradykinin which was fromSigma-Aldrich, St. Louis, Mo.):

1.Heparin - Binding Peptide V [Trp-Gln-Pro-Pro-Arg-Ala-Arg-Ile]; 1023 Da2. [D - Phe7]- Bradykinin [Arg-Pro-Pro-Gly-Phe-Ser-D-Phe-Phe-Arg]; 1111 Da 3.Bradykinin [Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg]; 1060 Da

Binding of PC-Biotin Peptide Mass Tags to Dual Affinity Beads

Performed as in Example 6 except that 150 μL of PC-Biotin peptide masstag solution, at a concentration of 5 pmoles/μL (750 pmoles peptide masstag), was added to a 1 μL bead pellet volume (30,000 beads); for a finaladded amount of biotin-labeled peptide mass tag of 25 fmoles per bead.As in Example 6, this step is to capture the PC-Biotin labeled peptidemass tags on the beads by way of the NeutrAvidin coating on the beads(peptide capture efficiency not measured). In this Example, 3 separatebatches of beads were prepared, each batch uniquely loaded with 1 of the3 aforementioned peptide species, creating 3 pure populations of uniquemass tag encoded beads. The Bradykinin mass tag was loaded onto theaforementioned “Marker Beads” (NeutrAvidin beads with direct fluorescentlabel attached to beads) while all other mass tags were loaded onto theaforementioned “Dual Affinity Beads” (NeutrAvidin beads additionallycontaining a common capture antibody for tagged recombinant proteins).

Binding of Recombinant Protein as “Bait” to Dual Affinity Beads

The following was performed on all bead species (kept separate) exceptthe “Marker Beads” which were not subjected to the procedures in theparagraph below:

Beads with the “Heparin-Binding Peptide V” identification tag wereloaded with cell-free expressed recombinant human p53 proteinessentially as in Example 6 with the following exceptions: Treatment ofthe beads with the cell-free expression reaction for recombinant p53protein capture was performed as in Example 6, except that immediatelyfollowing completion of the expression reaction (prior to mixing withbeads), protease inhibitor was additionally added to the expressionreaction to reduce the possibility of subsequent proteolytic degradationof the peptide mass tags on the beads (“Complete Mini Protease InhibitorCocktail Tablets” form Roche Applied Science, Indianapolis, Ind.,Catalog Number 11836153001; Stock Solution=1 mini-tablet in 1 mL ofpurified water; add 1/10 volume of Stock Solution to completed cell-freeprotein expression reactions). As in Example 6, the mechanism of captureof the cell-free expressed recombinant p53 protein on the Dual AffinityBeads is by way of the anti-HSV antibody coating on the beads and theC-terminal HSV epitope tag present in the recombinant p53. Finally,after loading the recombinant p53 protein onto the “Heparin-BindingPeptide V” encoded beads, washing was 4×400 μL, briefly with TBS-T.Beads with the “D-Phe7]-Bradykinin” identification tag were alsosubjected to the same above procedure, except that a blank cell-freeexpression reaction was used as a negative control instead of a p53cell-free expression reaction. In this case, the cell-free expressionreaction lacked the cognate expressible p53 DNA.

Pooling Beads and Fluorescence Detection of p53 Beads

All bead species, except “Marker Beads” which were added later, werethen pooled in equal numbers (2 species). The pooled beads were thenprobed with a fluorescently labeled anti-p53 antibody to specificallydetect the p53 containing beads. The fluorescently labeled anti-p53antibody was prepared in the same manner as the fluorescently labeledNeutrAvidin described in Example 7, except that the anti-p53 antibody(clone BP53-12, Santa Cruz Biotechnology, Santa Cruz, Calif.) was usedat 0.2 mg/mL in PBS and the Alexa Fluor® 594 SSE labeling reagent(Invitrogen, Carlsbad, Calif.) was used. The anti-p53 fluorescenceantibody probing was performed similar to as in Example 3 in the sectionheaded “Verification of Bound Recombinant Proteins”. After fluorescenceprobing, “Marker Beads” were spiked in at an approximate equal ratio tothe other bead species.

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Performed in the pico-well plates as in Examples 5 & 7.

Fluorescence Imaging of Pico-Well Plates

After MALDI-TOF mass-imaging, the pico-well plates were fluorescentlyimaged using a GenePix 4200 laser based microarray scanner (MolecularDevices, Sunnyvale, Calif.). This is possible because the fluorescentantibody probe is not significantly depleted during the MALDI-TOFprocess. However, fluorescence imaging could be achieved either beforeor after MALDI-TOF mass-imaging with comparable results. Furthermore,pica-well plates could be imaged from the bottom (through fiber optics)or from the top, with comparable results, despite the presence of MALDImatrix crystals on the top surface of the plate. Finally, fluorescenceimaging before matrix application and before MALDI-TOF was alsopossible, again with similar results.

Results:

The same region of the pico-well plate was imaged by both MALDI-TOF andfluorescence. Results are shown in FIG. 16. In FIG. 16A, a 2-colorfluorescence image is shown. Red spots are the “Marker Beads” and yellowspots are the p53 positive beads as detected with the anti-p53 antibodyprobe. Negative control blank beads are not visible in this image(separate fluorescence images of p53 positive and negative beads show asignal-to-noise ratio of >100:1; negative beads are visualized at veryhigh image contrast settings, by way of weak non-specific fluorescenceand auto-fluorescence background; image not shown). FIG. 16B is acolor-coded MALDI-TOF mass image of the same region, showing thelocalization of the 3 different photocleavable mass tags in the array(based on their respective m/z). In FIG. 16B, blue is the “Bradykinin”mass tag which encodes the “Marker Beads”, green the “Heparin-BindingPeptide V” mass tag which encodes the p53 beads and red the“[D-Phe7]-Bradykinin” mass tag which encodes the negative control blankbeads. This data shows distinct spots corresponding to beads carryingunique photocleavable mass tags, and little spatial overlap between the3 different mass tags in this experiment.

FIGS. 16C-E are direct (i.e. not offset) overlays of fluorescence imagesand MALDI-TOF mass images. Alignment of MALDI-TOF and fluorescenceimages was always based on the fluorescence and mass signals arisingfrom the “Marker Beads”. FIG. 16C is an overlay of the fluorescence fromthe “Marker Beads” (red) and the mass tag encoding those beads (blue).As seen, there is near 100% concordance between the fluorescence andMALDI-TOF images of the beads (overlapping colors appear as pink spots).FIG. 16D shows a sub-region (yellow boxed region from FIG. 16C) andcorresponds to overlaid fluorescence and MALDI-TOF images from both the“Marker Beads” and the p53 beads. Fluorescence arising from “MarkerBeads” is again shown in red, and the mass tag encoding those beads isshown in blue (overlap again observed as pink). Fluorescence arisingfrom the anti-p53 antibody probe (p53 positive beads) is again shown inyellow and the mass tag encoding those beads shown in green. In thisimage, 100% correlation between the “Marker Bead” fluorescence and thecorresponding mass tag encoding those beads is again observed. Likewise,excellent concordance is observed between the p53 positive beads asdetected by florescence (yellow) and the corresponding mass tag encodingthose beads (green). Of approximately 30 distinguishable p53 beads inthis field of view, only a 3-4 false negative beads are observed, thatwere detected by fluorescence but not by MALDI-TOF mass imaging. Notethat in some cases the green color (mass tag) masks the yellow(fluorescence) in the presented image, and that there are no falsepositive p53 beads, that is, beads detected with the mass tag but notfluorescence. In FIG. 16E, overlaid fluorescence and MALDI-TOF imagesare shown for the anti-p53 fluorescent antibody probe (yellow) and themass tag encoding the negative control blank beads (red), for the samesub-region as in FIG. 16D (“Marker Beads” again shown as pink due totheir respective overlapping colors). As expected, while the “MarkerBead” images correlate as before, there is poor overlap between theanti-p53 fluorescence (yellow) and the mass tag encoding the negativecontrol blank beads (red) which lack recombinant p53.

Taken together, these data show strong concordance between the mass tagencoding the p53 beads in the MALDI-TOF mass image and the fluorescenceimage of the anti-p53 antibody probe, and that this concordance isnon-random (due to lack of overlap from “Marker Beads” and negativecontrol blank beads).

Example 17 MALDI-TOF Mass-Imaging of Multiple Unique Photocleavable andNon-Cleavable Mass-Tag Encoded Bead Species in an Array: SynchronizingMass-Image for Bead Identification with Fluorescence Antibody Detectionof a Bead-Bound Bait Protein

In this Example, 10 distinct species of beads, each carrying a uniquepeptide mass tag, and 1 of the 10 additionally carrying boundrecombinant p53 protein (“bait”), were prepared separately, pooled andthen probed with a fluorescently labeled antibody (“prey”) to detect therecombinant p53 protein. An 11^(th) bead species was then spiked in as“Marker Beads”. These Marker Beads carried a unique peptide mass tag foridentification and the bead surface was covalently labeled with afluorophore having different and distinguishable spectral propertiesthan that used on the antibody probe. The pool of 11 bead species wasrandomly arrayed in the pica-well plates and then imaged by mass and byfluorescence.

Finally, it should be noted that 10 of the 11 peptide mass-tags usedwere attached to the beads by a photocleavable biotin (including on p53beads and Marker Beads), while the 11^(th) was attached via a modifiedreduced-affinity non-cleavable biotin, in order to additionallydemonstrate the possibility of using non-cleavable affinity-bound masstags.

Preparation of NeutrAvidin Coated Beads

NeutrAvidin coated 34 micron agarose beads were prepared as in Example5. As described below, these NeutrAvidin beads were used to prepare boththe “Dual Affinity Beads” and the “Marker Beads”.

Preparation of Dual Affinity Beads Coated with NeutrAvidin Directly andIndirectly with the Anti-HSV Tag Capture Antibody

The aforementioned NeutrAvidin beads were then loaded a biotin labeledanti-HSV tag polyclonal antibody as follows: Goat anti-HSV tagpolyclonal antibody was purchased from Bethyl Laboratories (Montgomery,Tex.), provided at 1 mg/mL in PBS. 800 μL of this antibody solution (800μg) was then mixed with 1/9^(th) volume of 1M sodium bicarbonate. Theantibody was biotin labeled by adding a 10-fold molar excess ofEZ-Link-Sulfo-NHS-LC-Biotin (Thermo-Fisher-Pierce, Rockford, Ill.) andreacting for 30 min with gentle mixing. The reaction was quenched byadding 1/9^(th) volume of 1 M glycine for 15 min with gentle mixing. Thelabeled antibody was then purified on a PD MidiTrap G-25 desaltingcolumn against TBS (see Example 3 for buffer) and according to themanufacturer's instructions (GE Healthcare Life Sciences, Piscataway,N.J.). The purified and biotin-labeled antibody was then diluted to 0.15μg/μL in TBS-T (see Example 3 for buffer). Using this solution, theNeutrAvidin beads were coated at a ratio of 12 μg of the biotin labeledanti-HSV tag antibody per each 1 μL of actual bead pellet volume for 30min with gentle mixing. Beads were then washed 4× briefly with an excessof TBS-T using the aforementioned Filtration Devices (see Example 3 fordevices). Beads were stored as a 20% (v/v) suspension at +4° C.

Note that because the antibody does not saturate all the biotin bindingsites on the NeutrAvidin beads, it was therefore possible toadditionally load biotin labeled peptide mass tags onto the beads (seelater steps in this Example).

Preparation of “Marker Beads” Coated with NeutrAvidin Directly andLabeled with Fluorescence

Performed as in Example 6 except that the aforementioned NeutrAvidinbeads (no bound antibody) were used for fluorescence labeling and theCy5-NHS activated (primary amine reactive) fluorescent dye labelingreagent was used (GE Healthcare Life Sciences, Piscataway, N.J.).

Preparation of Photocleavable (PC) and Non-Cleavable Biotin LabeledPeptide Mass Tags

Performed as in Example 5 except that the following commerciallyavailable peptides were used for labeling (peptide sequence in brackets)(all peptides purchased for labeling were from AnaSpec, Fremont, Calif.,except for Bradykinin which was from Sigma-Aldrich, St. Louis, Mo.):

1.Heparin - Binding Peptide V [Trp-Gln-Pro-Pro-Arg-Ala-Arg-Ile]; 1023 Da2.Alpha - Bag Cell Peptide (1 - 9) [Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu]; 1122 Da3. [D - Phe7]- Bradykinin [Arg-Pro-Pro-Gly-Phe-Ser-D-Phe-Phe-Arg]; 1111 Da 4.Antioxidant Peptide B [Thr-Arg-Asn-Tyr-Tyr-Val-Arg-Ala-Val-Leu]; 1254 Da5. Beta - Casomorphin (1 - 6), Bovine [Tyr-Pro-Phe-Pro-Gly-Pro]; 676 Da6. [Leu8, Des - Arg9]- Bradykinin [Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu]; 870 Da 7. [Ile - Ser]- Bradykinin (T - Kinin) [Ile-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg]; 1260 Da8. [Des - Arg1] - Bradykinin [Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg]; 905 Da9. LRRASLG [Leu-Arg-Arg-Ala-Ser-Leu-Gly]; 772 Da 10.Thrombin Receptor (42 - 48) Agonist, Human [Ser-Phe-Leu-Leu-Arg-Asn-Pro]; 846 Da11. Bradykinin [Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg]; 1060 Da

All above peptides were labeled on their N-terminus with theamine-reactive PC-Biotin-NHS reagent, as detailed in Example 5, exceptfor “Alpha-Bag Cell Peptide (1-9)”, which was labeled with theamine-reactive DSB-XTM Biotin SSE labeling reagent (Invitrogen,Carlsbad, Calif.) using the same protocol as in Example 5. DSB-XTMBiotin is a modified biotin derivative that has reduced affinity for(strept)avidin, believed to be several orders of magnitude weaker, andtherefore can be dissociated from (strept)avidin under comparativelymild conditions [Hirsch, Eslamizar, Filanoski, Malekzadeh, Haugland andBeechem (2002) Anal Biochem 308: 343-57]. While the native biotin moiety(ring structure) of the PC-Biotin labeled peptide mass tags is poorlydissociated from NeutrAvidin beads by the MALDI-TOF process (see Example5), it was expected that the lower affinity binding of DSB-XTM Biotinwould allow efficient dissociation of the mass tags via the denaturingMALDI-TOF matrix solution and/or by the energy introduced by the MALDIlaser.

Binding of PC-Biotin Peptide Mass Tags to Dual Affinity Beads

Performed as in Example 6 except that 150 μL of PC-Biotin or DSB-XTMBiotin peptide mass tag solution, at a concentration of 5 pmoles/μL (750pmoles peptide mass tag), was added to a 1 μL bead pellet volume (30,000beads); for a final added amount of biotin-labeled peptide mass tag of25 fmoles per bead. As in Example 6, this step is to capture thebiotin-labeled peptide mass tags on the beads by way of the NeutrAvidincoating on the beads (peptide capture efficiency not measured). In thisExample, 11 separate batches of beads were prepared, each batch uniquelyloaded with 1 of the 11 aforementioned peptide species, creating 11 purepopulations of unique mass tag encoded beads. The Bradykinin mass tagwas loaded onto the aforementioned “Marker Beads” (NeutrAvidin beadswith direct fluorescent label attached to beads) while all other masstags were loaded onto the aforementioned “Dual Affinity Beads”(NeutrAvidin beads additionally containing a common capture antibody fortagged recombinant proteins).

Binding of Recombinant Protein as “Bait” to Dual Affinity Beads

Although in some embodiments all mass tagged bead species canadditionally carry a “bait” molecule such as a protein, in this Example,with the exception of the “Alpha-Bag Cell Peptide (1-9)” and“Heparin-Binding Peptide V” mass tag encoded beads, the other 10 masstagged bead species were not loaded with cell-free expressed recombinantprotein and thus not subjected to this portion of the procedure:

Beads with the “Alpha-Bag Cell Peptide (1-9)” identification tag wereloaded with cell-free expressed recombinant human p53 proteinessentially as in Example 6 with the following exceptions: Treatment ofthe beads with the cell-free expression reaction for recombinant p53protein capture was performed as in Example 6, except that immediatelyfollowing completion of the expression reaction (prior to mixing withbeads), protease inhibitor was additionally added to the expressionreaction to reduce the possibility of subsequent proteolytic degradationof the peptide mass tags on the beads (“Complete Mini Protease InhibitorCocktail Tablets” form Roche Applied Science, Indianapolis, Ind.,Catalog Number 11836153001; Stock Solution=1 mini-tablet in 1 mL ofpurified water; add 1/10 volume of Stock Solution to completed cell-freeprotein expression reactions). As in Example 6, the mechanism of captureof the cell-free expressed recombinant p53 protein on the Dual AffinityBeads is by way of the anti-HSV antibody coating on the beads and theC-terminal HSV epitope tag present in the recombinant p53. Finally,after loading the recombinant p53 protein onto the “Alpha-Bag CellPeptide (1-9)” encoded beads, washing was 4×400 μL briefly with TBS-T.Beads with the “Heparin-Binding Peptide V” identification tag were alsosubjected to the same above procedure, except that a blank cell-freeexpression reaction was used as a negative control instead of a p53cell-free expression reaction. In this case, the cell-free expressionreaction lacked the cognate expressible p53 DNA.

Pooling Beads and Fluorescence Detection of p53 Beads

All bead species, except “Marker Beads” which were added later, werethen pooled in equal numbers (10 species). The pooled beads were thenprobed with a fluorescently labeled anti-p53 antibody to specificallydetect the p53 containing beads. The fluorescently labeled anti-p53antibody was prepared in the same manner as the fluorescently labeledNeutrAvidin described in Example 7, except that the anti-p53 antibody(clone BP53-12, Santa Cruz Biotechnology, Santa Cruz, Calif.) was usedat 0.2 mg/mL in PBS and the Alexa Fluor® 594 SSE labeling reagent(Invitrogen, Carlsbad, Calif.) was used. The anti-p53 fluorescenceantibody probing was performed similar to as in Example 3 in the sectionheaded “Verification of Bound Recombinant Proteins”. After fluorescenceprobing, “Marker Beads” were spiked in at an approximate equal ratio tothe other bead species.

Mass Tag Photocleavage and MALDI-TOF Mass Spectrometry Imaging of Beads

Performed in the pico-well plates as in Examples 5 & 7.

Fluorescence Imaging of Pico-Well Plates

After MALDI-TOF mass-imaging, the pico-well plates were fluorescentlyimaged using a GenePix 4200 laser based microarray scanner (MolecularDevices, Sunnyvale, Calif.). This is possible because the fluorescentantibody probe is not significantly depleted during the MALDI-TOFprocess. However, fluorescence imaging could be achieved either beforeor after MALDI-TOF mass-imaging with comparable results. Furthermore,pico-well plates could be imaged from the bottom (through fiber optics)or from the top, with comparable results, despite the presence of MALDImatrix crystals on the top surface of the plate. Finally, fluorescenceimaging before matrix application and before MALDI-TOF was alsopossible, again with similar results.

Results:

The same 4.8×2.8 mm region of the pico-well plate was imaged by bothMALDI-TOF and fluorescence (13.44 mm²; roughly 6,000 wells). Results areshown in FIG. 17. In FIG. 17A, image processing was as follows: Separategrayscale mass-images, corresponding to the m/z for each of the masstags, were processed using the public domain scientific imaging softwareImageJ v1.42q [Rasband, W. S., ImageJ, U.S. National Institutes ofHealth, Bethesda, Md., USA, http://rsb.info.nih.gov/ij/, 1997-2009;Abramoff, M. D., Magelhaes, P. J., Ram, S. J. “Image Processing withImageJ”. Biophotonics International, volume 11, issue 7, pp. 36-42,2004]. First, a 2 pixel median filter was applied for noise reductionand then the “Find Edges” algorithm was applied to trace the outlines ofindividually resolvable “features” (spots) in the images. The resultantimages of the spot outlines for each mass tag were then overlaid.Overlapping (intersecting) spot outlines are shown in white in FIG. 17A.In the region scanned and shown in FIG. 17A, all mass tags except the“LRRASLG” peptide (not detected) are represented at least once asindividually resolvable non-overlapping spots.

In FIG. 17B, a 5-color hybrid image was created by overlaying thecolorized grayscale mass-images of 3 selected mass-tags with thefluorescence images of both the anti-p53 antibody probe and the “MarkerBead” label. Note that the fluorescence images were offset from the massimages to allow visualization of both mass tags and fluorescence arisingfrom the same beads. Fluorescence and mass images were aligned based onthe “Marker Beads”.

Data in FIG. 17B show that p53 beads detected with the anti-p53fluorescent antibody (yellow) show excellent concordance with the masstag encoding the p53 beads (green). 4 of 5 spots are concordant (80%),with one false negative spot detected by fluorescence and not MALDI-TOF(marked with white “X” in FIG. 17B). Conversely, spots identified as theminus p53 negative control beads by virtue of their cognate mass tag(purple), show no concordance with the anti-p53 fluorescence (yellow),as expected. Taken together, these data show that the concordancebetween the anti-p53 fluorescent antibody probe and the mass tagencoding (identifying) the p53 beads, is not by random chance Likewise,the fluorescence of the “Marker Beads” (red spots with white center)show 100% concordance with their cognate mass tag (light blue).

Finally, comparison of florescence spot sizes with the MALDI-TOF imagesof both the “Marker Beads” and p53 beads suggests that the spatialresolution of the MALDI-TOF imaging approaches that of the fluorescenceimaging (fluorescence scanned at 10 micron/pixel resolution).

Example 18 Colorimetric Detection of Beads in Pico-Well Plates

In this Example, the ability to detect 34 micron diameter beads in thewells of pico-well plates is shown. In this case, the aforementioned 34micron diameter agarose beads were coated with streptavidin-HRP andincubated in a precipitating chromogenic HRP substrate (TrueBluePeroxidase Substrate; KPL Inc., Gaithersburg, Md.). The now opaquelycolored beads were deposited into the wells of commercially obtainedpico-well plates (PicoTiter™ Plates designed for 454 Life Sciences GSFLX DNA Sequencer; Roche Applied Science, Indianapolis, Ind.), ThePicoTiter™ Plates are similar to the pico-well plates used in previousExamples, except that the slide (plate) thickness is 2 mm instead of 1mm. Opaquely colored beads were imaged in the wells of the PicoTiter™Plates using a visible light based ArrayIt SpotWare™ FlatbedColorimetric Microarray Scanner (TeleChem International, Inc. ArrayIt™Division, Sunnyvale, Calif.). Results shown in FIG. 18 clearlydemonstrate that the beads can be imaged colorimetrically using visiblelight based techniques (e.g. CCD based visible light imager). In thegrayscale image in FIG. 18, wells containing the beads are identified bytheir dark color (dark spots), whereas empty wells are observed as clear(white spots).

In some embodiments of the technology described in this patent,colorimetric detection of the beads could be used to visualize probe or“prey” binding to “bait” molecules on the beads. Alternatively,colorimetrically detected beads could serve as “landmarks” to direct theMALDI-TOF instrument to specific wells or coordinates in the array.

FIGURES AND TABLES Description of the Figures for Experimental Examples

FIG. 1. Affinity Purification of Peptides onto an Agarose Bead ResinFollowed by Mass Spectrometry Detection from Single Beads. Singleagarose beads approximately 75-150 microns in diameter carrying a testpeptide immobilized by a bead-bound antibody directed against thepeptide's N-terminal FLAG epitope tag were manually selected, depositedon a suitable substrate and scanned using the laser beam of a MALDI-TOFmass spectrometer. The labels in parenthesis correspond to the rawsignal intensity of the expected target peak (arbitrary units). Theasterisks indicate the minor matrix adduct of the target peak. Spectrafrom 3 different individual agarose beads are shown.

FIG. 2. Detection and Mass-Imaging of Different Populations of Peptideson Individual Beads Using Scanning MALDI-TOF Mass Spectrometry. Agarosebeads approximately 75-150 microns in diameter, each bead carrying oneof two possible test peptides immobilized by a bead-bound antibodydirected against their common N-terminal FLAG epitope tag, weredeposited on a suitable substrate and the substrate scanned using thelaser beam of a MALDI-TOF mass spectrometer. The image (left) is atwo-color overlay of the two mass-images of the beads, created usingspectral intensity at the m/z (mass/charge) corresponding to themolecular weight of the test peptides. Sample spectra (right) areprovided from single beads showing that the beads carry a homogeneouspopulation of one peptide.

FIG. 3. Single-Bead MALDI-TOF Mass Spectrometry Mass-Imaging of UniqueMass Tags on 34 Micron Agarose Beads Also Carrying Recombinant Proteins.(A.) (top) Color-coded overlaid MALDI-TOF mass spectrometry mass-imagesof three unique HSV peptide mass tags on individual protein-beads (toppanel). Beads are deposited in specialized pico-well plates (substrate),whose dimensions restrict loading to 1 bead/well, prior to MALDI-TOFmass spectrometry mass-imaging of the substrate. Blue=“Blank” beadslacking recombinant protein but containing 1,368 Da version of HSVpeptide mass tag; Green=human p53 recombinant protein beads containing2,048 Da version of HSV peptide mass tag; Red=human KLHL recombinantprotein beads containing 2,074 Da version of HSV peptide mass tag.Sample spectra (color-coded) are shown for two of the mass tags asdetected from single beads (bottom). (B.) Separately, fluorescenceprobing and imaging of an aliquot of beads was performed using afluorescently labeled (Cy3) anti-VSV-G tag antibody to verify therecombinant proteins are indeed present, by virtue of this commonN-terminal epitope tag in all recombinant proteins. “Blank” correspondsto beads processed in the same manner but lacking recombinant protein.Inset “High Contrast” box shows presence of beads in the “Blank” usinghigh contrast image settings.

FIG. 4. Synchronization of Fluorescence Image and Mass SpectrometryMass-Image of Individually Resolved 34 Micron Agarose Beads in Pico-WellPlates. Two-color overlay. Red=MALDI-TOF mass spectrometry mass-image ofan HSV peptide mass tag on beads in a pica-well plate;Green=fluorescence image of peptide mass tag on same beads in sameregion of pico-well plate. The two images were intentionally offset toshow spot concordance.

FIG. 5. MALDI-TOF Mass Spectrometry Mass-Imaging of Photocleavable MassTags on Individual 34 Micron Agarose Beads in Pico-Well Plates. [Top]Diagrammatic representation of the experimental design. [Lower Right]MALDI-TOF mass spectrometry mass-image of the photocleaved PC-BiotinVSV-G mass tag peptide from individual beads in the pico-well plate. Thebeads in the pico-well plate were pre-treated with near-UV light (+UV)to photo-release the mass tag peptide before MALDI-TOF imaging. Asection of the plate was masked (−UV) as a negative control. The variousexperimental permutations performed included with and without thepeptide mass tag on the beads (“+Mass Tag” and “−Mass Tag” respectively)and with and without light pre-treatment of the beads (+UV and −UVrespectively). A mass-image for the “−Mass Tag +UV” experimentalpermutation is not shown. [Lower Left] Representative mass spectra areshown from individual beads for all three experimental permutationsperformed.

FIG. 6. Photocleavable Mass Tags (for Bead Identification) Co-Loadedwith “Bait” Molecules for Multiplex Bioassays: “Bait” Detection and MassSpectrometry Readout from Beads. (A.) Bead-ELISA results for detectionof bead-bound human recombinant p53 “bait” from an entire population ofbeads. Detection of bead-bound p53 was by virtue of an anti-VSV-G tagantibody conjugated to an enzymatic reporter (chemiluminescencereadout); the anti-VSV-G tag antibody binds this epitope tag present inthe p53 protein. The p53 signal (i.e. anti-VSV-G epitope tag antibodysignal) was normalized to the relative bead amount in each sample(“Normalized p53 Signal”) using a separate fluorescence tag conjugatedto the bead surface. On the X-axis of the graph, the presence (+) orabsence (−) of recombinant human p53 on the beads is indicated, as wellas the presence or absence of the PC-Biotin conjugated bradykininpeptide mass tag (“Bradykinin PC-Mass Tag”). (B.) MALDI-TOF massspectrometry measurement of the PC-Biotin conjugated bradykinin peptidemass tag (“Bradykinin PC-Mass Tag”) (1,060 Da) following photo-releasefrom an aliquot of the same batch of beads.

FIG. 7. Mass-Tagged Probes for MALDI-TOF Mass Spectrometry Mass-Imagingof Individually Resolved Beads: Detection of Serum Autoantibody Againsta Bead-Bound Autoantigen by Fluorescence and MALDI-TOF Mass Spectrometryin Pico-Well Plates. The top panel is a diagrammatic representation ofthe experimental design and the bottom panel is the results. Bothfluorescence and MALDI-TOF mass-imaging detect the bound autoantibody.Beads either contained (+) or lacked (−) the human recombinantautoantigen (“Ag.”) and were treated with either anautoantibody-positive patient serum (PBC; Primary Biliary Cirrhosisautoimmune serum) or an autoantibody-negative normal patient serum(“Norm”). The fluorescence (Cy3) image (lower left) was intentionallyset to a saturating contrast to show the presence of beads in allsamples, including the negative controls (by weak non-specificfluorescence). MALDI-TOF mass spectrometry mass-imaging (lower right)was used to detect the photo-released PC-Biotin conjugated VSV-G peptidemass tag from the bound anti-[human IgG] secondary antibody probe on thebeads, for autoantibody readout. Sample spectra from individual beadsare shown in addition to the mass-image. Note that fluorescence imagesand mass-images are of different pico-well plates (same batch of beads)and hence not synchronized in this case.

FIG. 8. Application of the Protease Enzyme to the Bead Library Depositedon the Pico-Well Plates: Efficient Protein Digestion without the Loss ofSpatial Resolution. Fluorescence scan of the fluorescent Cy3-labeledbead array following trypsin digestion and MALDI matrix depositionmeasured from the bottom of pico-wells (top image) and the slide surface(bottom image). Inset: region of the MALDI spectrum showing a peakarising from the trypsin digest fragment of p53.

FIG. 9. Measurement of Changes in the Protein Concentration Using aCombination of Protein Isotope Labeling, Proteolytic Digestion andMALDI-TOF Mass Spectrometry Mass-Imaging Analysis of Bead Microarrays.Panel A: overlay of two mass spectra of pure populations of beadscarrying either non-labeled or ¹³C₆-Leu-labeled p53 after trypsindigestion showing a mass-shifted peak due to the Leucine incorporation.Panel B: a mass-spectrum in the same region as Panel A obtained afternon-labeled and ¹³C₆-Leu-labeled p53 were mixed in a 5:1 ratio prior tothe bead binding. Panel C: MALDI MSI scan of a bead array containing twopopulations of beads each carrying either non-labeled or¹³C₆-Leu-labeled p53 after the trypsin digestion. Red spots are areasthat exhibit a 1006 Da peak and green spots exhibit a 1018 Da peak. Thedotted white line indicated the area of the slide where beads weredeposited.

FIG. 10. Decoding of DNA Tags Photo-Released from Beads and Analyzed ona Massively Parallel RT-PCR Chip. DNA-Tags in this case were human geneORFs. The red numbers above the bar indicate the equivalent number ofbeads analyzed by the RT-PCR chip.

FIG. 11. Physical Pre-Selection of Beads for Decoding using aFluorescence Activated Cell-Sorting (FACS) Instrument. Blank beads(control) and autoantigen beads were probed with a positive autoimmuneserum and autoantibody detected with a fluorescent (fluorescein)secondary antibody. Beads were the same 34 micron diameter agarose beadsused extensively in previous Examples for MALDI-TOF mass spectrometrymass-imaging. The x-axes of the graphs are the channel for detection ofautoantibody binding (fluorescein) and the y-axes a control (irrelevant)fluorescence channel.

FIGS. 12A-C. Immune Response Profiling on Beads: Photocleavable (PC)Mass-Tagging and MALDI-TOF Mass-Imaging of the Individually ResolvedBeads in an Array. (A.) Schematic of basic experimental design.PC-Mass-Tag encoded beads (“Bead Identification Tag”), carrying eitherthe SmB protein autoantigen or GST A2 as a negative control protein,were probed with a human SLE serum to detect autoantibody binding. Theautoantibody probe (“Probe Tag”) was also detected with a uniquePC-Mass-Tag. (B.) MALDI-TOF mass-image of PC-Mass-Tags from individuallyresolved beads in an array: (1) Offset image of SmB Bead IdentificationTag (green) as well as the autoantibody Probe Tag (red). (2) Same regionof the array viewed as 3-color direct mass-image overlay, with the BeadIdentification Tag for GST additionally shown in white/gray;co-localization of the green and red mass tag signals from SmB beadsshows as yellow spots (not offset). (C.) ELISA analysis on the same SLEserum to validate the results from the bead-based mass-imaging assay.Detection is shown for the “Autoantibody” as well as the “VSV-G Tag”(common tag in both expressed proteins; SmB and GST).

FIGS. 13A-C. MALDI-TOF Mass-Imaging of 10 Distinct Peptide-Bead Speciesin an Array in Conjunction with Antibody Detection of “Bait” Molecules.(A.) Mass-image of bead array with all 10 peptide species colored red.Beads are 34 microns in diameter. Regions of overlap of any 2 peptidespecies are shown in yellow. (B.) Mass-image of same region of beadarray with each panel showing an image of 1 of the 10 specific peptidespecies (each panel is different peptide species). (C.) Fluorescencebead image. An aliquot of the same beads was separately imaged byfluorescence to detect the bound antibody probe (“prey”) directedagainst an epitope tag in the recombinant p53 protein (“bait”). Purepopulations of “Blank” beads (beads loaded with a blank cell-freeexpression reaction) and “p53” beads (beads loaded with a p53 cell-freeexpression reaction) were included as controls. The pooled beadscorrespond to all 10 bead species including 1 species of uniquelymass-tagged p53 beads and 9 species of uniquely mass-tagged non-p53beads (no recombinant protein). A “Higher Contrast Image” allows forbetter visualization of the non-p53 beads, detectible by weaknon-specific background fluorescence.

FIGS. 14A-B. Methods of Direct Attachment of Photocleavable Mass Tags toSurfaces Such as Beads. (A.) Example structure of an NHS-activatedprotected-amine linker used to modify the N-terminal of peptide masstags. The compound (“Photocleavable Amine Linker”) has an active NHSester on one end and a protected amine on the other, with a “Spacer” armand a photocleavable nucleus (“P”) in the center. Note that while ahydrocarbon “Spacer” arm is shown, a multitude of possible structuresand lengths can be used. Following peptide modification, the protectinggroup, e.g. an acid labile group, can be removed to “De-Protect” theamine on the linker. This generates a peptide modified to have aphotocleavable primary amine group at its N-terminus (“PC-AminePeptide”). (B.) This photocleavable primary amine group on the peptidecan be reacted with NHS activated beads for example (peptide would lackany other free primary amines), thus creating peptides photocleavablylinked to the bead surface via a direct covalent attachment. Uponillumination with the proper light (“hv”), the peptide is photo-releasedfrom the bead for analysis by mass spectrometry.

FIGS. 15A-B. Mass Spectrometry Readout and Mass-Imaging fromIndividually Resolved 34 Micron Beads in Metal Coated Pico-Well Plates.(A) MALDI-TOF mass-image of bradykinin mass-tag and fluorescence imageof anti-p53 antibody probe from beads in the two types of pico-wellplates (plain glass and gold-coated). (B) Comparison of MALDI-TOFspectra from in situ trypsinized human recombinant ACVR-2B from beads inthe plain glass and gold-coated pico-well plates. Red labels are peptidefragments that could be correctly assigned to ACVR-2B protein sequencebased on mass. The light green line and label indicates the mass abovewhich little to no detectible peptides are observed on the glasspico-well plates.

FIGS. 16A-E. MALDI-TOF Mass-Imaging of Multiple Unique PhotocleavableMass-Tag Encoded Bead Species in an Array: Synchronizing Mass-Image forBead Identification with Fluorescence Antibody Detection of a Bead-BoundBait Protein All images shown are direct (i.e. not offset) overlays.(A.) 2-color fluorescence image. Red spots are the “Marker Beads” andyellow spots are the p53 positive beads as detected with the anti-p53antibody probe. Negative control blank beads are present but not visiblein this image. (B.) Color-coded MALDI-TOF mass image of the same region,showing the localization of the 3 different photocleavable mass tags inthe array (based on their respective m/z). Blue is the “Bradykinin” masstag which encodes the “Marker Beads”, green the “Heparin-Binding PeptideV” mass tag which encodes the p53 beads and red the“[D-Phe7]-Bradykinin” mass tag which encodes the negative control blankbeads. (C-E) Direct (i.e. not offset) overlays of fluorescence imagesand MALDI-TOF mass images. Alignment of MALDI-TOF and fluorescenceimages was always based on the fluorescence and mass signals arisingfrom the “Marker Beads”. (C) Overlay of the fluorescence from the“Marker Beads” (red) and the mass tag encoding those beads (blue) forthe same region of the array (overlapping colors appear as pink spots).(D) Sub-region of the array (yellow boxed region from panel C).Corresponds to overlaid fluorescence and MALDI-TOF images from both the“Marker Beads” and the p53 beads. Fluorescence arising from “MarkerBeads” is again shown in red, and the mass tag encoding those beads isshown in blue (overlap again observed as pink). Fluorescence arisingfrom the anti-p53 antibody probe (p53 positive beads) is again shown inyellow and the mass tag encoding those beads shown in green. (E.)Overlaid fluorescence and MALDI-TOF images are shown for the anti-p53fluorescent antibody probe (yellow) and the mass tag encoding thenegative control blank beads (red), for the same sub-region as in panelD (“Marker Beads” again shown as pink due to their respectiveoverlapping colors).

FIGS. 17A-B. MALDI-TOF Mass-Imaging of Multiple Unique Photocleavableand Non-Cleavable Mass-Tag Encoded Bead Species in an Array:Synchronizing Mass-Image for Bead Identification with FluorescenceAntibody Detection of a Bead-Bound Bait Protein (A) Overlay of spot(bead) outlines identified by mass-imaging of all mass tags in thisexperiment. Each individual “spot” (continuous shape) in the presentedimage corresponds to a single unique mass tag (10 of 11 mass tagsdetected as an individually resolved spot at least once; 1 mass tag notdetected). Overlapping (intersecting) spot outlines are shown in white.(B.) 5-color hybrid image created by overlaying the colorized grayscalemass-images of 3 selected mass-tags with the fluorescence images of boththe anti-p53 antibody probe and the “Marker Bead” label. The legenddenotes the color-coding in the presented image. Yellow=fluorescence ofanti-p53 antibody probe; Green=mass tag encoding p53 containing beads;Purple=mass tag encoding minus p53 negative control beads; Red (withwhite center)=fluorescence of “Marker Bead” label; Light Blue=mass tagencoding “Marker Beads”.

FIG. 18. Colorimetric Detection of Beads in Pico-Well Plates. Opaquelycolored beads were deposited into the wells of pico-well plates andimaged using a visible light based colorimetric microarray scanner. Inpresented grayscale image (2 fields of view shown), beads in the wellsare identified by their dark color (dark spots), whereas empty wellsappear as clear (white spots).

Description of the Figures for Specifications

FIG. S01. Proteomic-Wide Screening. A global proteome array provides apowerful means to obtain information about how specific proteins in theproteome interact with a variety of “inputs” such as other proteins,small molecules or complex clinical samples.

FIG. S02. Schematic Showing Two Typical Protein MicroarrayConfigurations. Top: An array of antibodies are deposited on biochipplanar surface which selectively capture specific analytes occur.Bottom: Various proteins (e.g. autoantigens) are dried on biochipsurface in order to probe molecular interactions such as specificantibody interaction. Typically, fluorescent labeling of captured orinteracting species is used for detection (read-out). Adapted fromhttp://www.elmat.lth.se/uploads/pics/Fig1.jpg.

FIG. S03. Example of Bead-Based Global Proteomic Screening (Bead-GPS)with MALDI-TOF MS and Fluorescence Readout.

FIG. S04. Photo-Release of Mass-Tags from Beads. Green mass-tag codesfor positive “hit” due to autoantibody interaction with protein bound tobead. Red mass-tag codes for protein identity on bead. Purple mass-tagcode is common to all beads in a library used to screen a particularblood serum sample. Yellow oval represents photocleavable linker whichis cleaved by near-UV light.

FIG. S05. Attachment of Photocleavable (PC) Mass-Tags to Beads. (1.)PC-Biotin (B) labeled mass-tag attached to (strept)avidin coating(green). (2.) PC-amine modified mass-tag covalently attached through NHSsurface chemistry. Yellow circles are the photocleavable nuclei.Affinity capture elements for immobilization of “bait” molecules (e.g.recombinant proteins) are also attached by NITS surface chemistry (notshown).

FIG. S06. Activated Photocleavable (PC)-Mass Tag Reagents for LabelingProbes Used to Query a Bead Library. NHS-activated (amine-reactive)peptide mass tags containing a photocleavable (PC) linker (yellow) aredirectly conjugated (red arrow) to antibody probes (green). The peptide(purple) lacks any free primary amines (blocked or absent) to preventself-reactivity.

FIG. S07. Individual steps involved in Bead-GPS.

FIG. S08. Expression Plasmids from the ORFeome. Plasmid constructs ofthe source ORFeome library (e.g. pDONR223) and conversion into thedestination vector (pVSV-DEST) used for cell-free protein expression.

FIG. S09. Dual-Chambered Automation Compatible Devices for High YieldWheat Germ CECF Expression. Devices use a 96-well frame and foot printfor automation compatibility.

FIG. S10. Yield Comparison for Expression of snRNP C in RabbitReticulocyte and Wheat Germ Cell-Free Systems. Analysis was byT²-ELISA™. To avoid saturation of the ELISA plate, the amount ofexpression reaction input into the ELISA was varied.

FIG. S11. Basic Configurations of Beads Comprising BS-LIVE-PRO.Cell-Free expressed proteins (purple) are in situ captured/purified ontobeads using an antibody against the C-terminal epitope tag (“C-Tag”).Bead-bound proteins are quantified by fluorescence (“F”)antibody-mediated detection of the N-terminal epitope tag (N-Tag). In analternative configurations, biotins (“B”) and/or fluorophores (“F”) aredirectly incorporated using tRNA-mediated co-translational labelingtechnology developed by AmberGen [Lim and Rothschild (2008) Anal Biochem383: 103-115]. This affords direct capture onto (strept)avidin beadsand/or detection by direct fluorescence.

FIG. S12. Normalization of Expressed Protein Level on Beads Based onT2-ELISA™. Based on estimates of expression yield by T²-ELISA™, theratio of beads used for capture was modulated to normalize for yielddifferences of 5 proteins.

FIG. S13. In Situ Trypsinization Followed by MALDI-MS Imaging andIdentification of Cell-Free Expressed Human p53 and GST A2 on IndividualBeads.

1. A method of detecting the interaction of prey molecules with baitmolecules on an array of beads, comprising a) providing a mixturecomprising first and second beads, said first bead comprising a firstmass tag and a first bait molecule, said second bead comprising a secondmass tag and a second bait molecule, wherein said first and second baitmolecules and said first and second mass tags are different; b)contacting said first and second bait molecules with a solutioncomprising a prey molecule, wherein said prey molecule comprises a thirdmass tag which is different than first and second mass tags; c) makingan array with said beads; and d) subjecting said array to mass specanalysis under conditions wherein binding of said prey molecule to saidbait molecule is detected by measuring mass tags.
 2. The method of claim1, wherein said mass tags are selected from the group consisting ofpolypeptides, nucleic acids, linear polymers, branched polymers.
 3. Themethod of claim 2, wherein said mass tags are released by light anddetected during the mass spectrometric analysis of step d).
 4. Themethod of claim 1, wherein said first and second mass tag are coupled tobead through a binding agent.
 5. The method of claim 4, wherein saidbinding agent is streptavidin.
 6. The method of claim 1, wherein saidprey molecule is an antibody.
 7. The method of claim 1, wherein saidprey molecule is a protein.
 8. The method of claim 1, wherein said preymolecule is a polypeptide.
 9. The method of claim 1, wherein said preymolecule is a nucleic acid.
 10. The method of claim 1, wherein said preymolecule is a small drug compound.
 11. The method of claim one whereinthird mass tag is coupled to prey molecule through a binding agent. 12.The method of claim 11 wherein binding agent is an antibody.
 13. Themethod of claim 11, wherein said antibody is fluorescently labeled. 14.The method of claim 1, wherein said mass spec analysis in step d)comprises measuring mass tags from individual beads.
 15. The method ofclaim 1, wherein said mass spec analysis in step d) comprises MALDI-MSimaging of individual beads in said array.
 16. The method of claim 1,wherein said array of step c) is imaged fluorescently prior to said massspec analysis of step d).
 17. The method of claim 1, wherein said arrayof step c) is imaged fluorescently subsequent to said mass spec analysisof step d).
 18. The method of claim 1, wherein said array of step c) isa random array.
 19. A method of detecting the interaction of preymolecules with bait molecules , comprising a) providing a mixturecomprising first and second beads, said first bead comprising a firstmass tag and a first bait molecule, said second bead comprising a secondmass tag and a second bait molecule, wherein said first and second baitmolecules and said first and second mass tags are different; b) makingan array with said beads; c) contacting said first and second baitmolecules with a solution comprising a prey molecule, wherein said preymolecule comprises a mass tag; and d) subjecting said array to MALDImass spec analysis under conditions wherein binding of said preymolecules to a bait molecule is detected.
 20. The method of claim 19,wherein said mass tags are selected from the group consisting ofpolypeptides, nucleic acids, linear polymers, branched polymers.
 21. Themethod of claim 20, wherein said mass tags are released by light anddetected during the mass spectrometric analysis of step d).
 22. Themethod of claim 19, wherein said first and second mass tag are coupledto bead through a binding agent.
 23. The method of claim 22, whereinsaid binding agent is streptavidin.
 24. The method of claim 19, whereinsaid prey molecule is an antibody.
 25. The method of claim 19, whereinsaid prey molecule is a protein.
 26. The method of claim 19, whereinsaid prey molecule is a polypeptide.
 27. The method of claim 19, whereinsaid prey molecule is a nucleic acid.
 28. The method of claim 19,wherein said prey molecule is a small drug compound.
 29. The method ofclaim one wherein third mass tag is coupled to prey molecule through abinding agent.
 30. The method of claim 29 wherein binding agent is anantibody.
 31. The method of claim 29, wherein said antibody isfluorescently labeled.
 32. The method of claim 19, wherein said massspec analysis in step d) comprises measuring mass tags from individualbeads.
 33. The method of claim 19, wherein said mass spec analysis instep d) comprises MALDI-MS imaging of individual beads in said array.34. The method of claim 19, wherein said array of step c) is imagedfluorescently prior to said mass spec analysis of step d).
 35. Themethod of claim 1, wherein said array of step c) is imaged fluorescentlysubsequent to said mass spec analysis of step d).
 36. The method ofclaim 19, wherein said array of step c) is a random array.